Diphenylmethyl compounds useful as muscarinic receptor antagonists

ABSTRACT

This invention provides compounds of formula I: 
     
       
         
         
             
             
         
       
     
     wherein a, b, c, e, m, n, Ar 1 , R 1 , R 2 , R 3 , R 4a , R 4b , R 5  and R 6  are as defined in the specification. The compounds of formula I are muscarinic receptor antagonists. The invention also provides pharmaceutical compositions containing such compounds, processes and intermediates for preparing such compounds and methods of using such compounds to treat pulmonary disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/552,401, filed on Mar. 11, 2004; the entire disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel diphenylmethyl compounds havingmuscarinic receptor antagonist or anticholinergic activity. Thisinvention also relates to pharmaceutical compositions comprising suchdiphenylmethyl compounds, processes and intermediates for preparing suchdiphenylmethyl compounds and methods of using such diphenylmethylcompounds to treat pulmonary disorders.

2. State of the Art

Pulmonary or respiratory disorders, such as chronic obstructivepulmonary disease (COPD) and asthma, afflict many millions of peopleworldwide and such disorders are a leading cause of morbidity andmortality.

Muscarinic receptor antagonists are known to provide bronchoprotectiveeffects and therefore, such compounds are useful for treatingrespiratory disorders, such as COPD and asthma. When used to treat suchdisorders, muscarinic receptor antagonists are typically administered byinhalation. However, even when administered by inhalation, a significantamount of the muscarinic receptor antagonist is often absorbed into thesystemic circulation resulting in systemic side effects, such as drymouth, mydriasis and cardiovascular side effects.

Additionally, many inhaled muscarinic receptor antagonists have arelatively short duration of action requiring that they be administeredseveral times per day. Such a multiple-daily dosing regime is not onlyinconvenient but also creates a significant risk of inadequate treatmentdue to patient non-compliance with the required frequent dosingschedule.

Accordingly, a need exists for new muscarinic receptor antagonists. Inparticular, a need exists for new muscarinic receptor antagonists thathaving high potency and reduced systemic side effects when administeredby inhalation. Additionally, a need exists for inhaled muscarinicreceptor antagonists having a long duration of action thereby allowingfor once-daily or even once-weekly dosing. Such compounds are expectedto be particularly effective for treating pulmonary disorders, such asCOPD and asthma, while reducing or eliminating side effects, such asdry-mouth and constipation.

SUMMARY OF THE INVENTION

The present invention provides novel diphenylmethyl compounds havingmuscarinic receptor antagonist or anticholinergic activity. Among otherproperties, compounds of this invention are expected to possess highpotency and reduced systemic side effects when administered byinhalation and to have a long duration of action.

Accordingly, in one of its composition aspects, this invention providesa compound of formula I:

wherein

each R¹ and R² are independently selected from (1-4C)alkyl,(2-4C)alkenyl, (2-4C)alkynyl, (3-6C)cycloalkyl, cyano, halo, —OR^(a),—SR^(a), —NR^(a)R^(b), —S(O)R^(c) and —S(O)₂R^(c); where each R^(a) andR^(b) independently represents hydrogen, (1-4C)alkyl, (2-4C)alkenyl,(2-4C)alkynyl or (3-6C)cycloalkyl; each R^(c) independently represents(1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl or (3-6C)cycloalkyl; or twoadjacent R¹ groups or two adjacent R² groups are joined together to form(3-6C)alkylene, (2-4C)alkylene-O— or —O-(2-4C)alkylene-O—;

a and b each independently are 0 or an integer of from 1 to 5;

each R³ independently is fluoro or (1-4C)alkyl;

c is 0 or an integer of from 1 to 3;

R^(4a) and R^(4b) are independently selected from hydrogen, (1-4C)alkyland phenyl-(1-4C)alkyl; or R^(4a) and R^(4b) together with the nitrogenatom to which they are attached form a (3-6C)heterocyclic ringoptionally containing one additional heteroatom selected from nitrogen,oxygen or sulfur and wherein the heterocyclic ring is unsubstituted orsubstituted with 1 or 2 substituents selected independently from(1-4C)alkyl and fluoro;

e is 1 or 2;

m is 1, 2, 3 or 4;

Ar¹ represents a phenylene group or a (3-5C)heteroarylene groupcontaining 1 or 2 heteroatoms selected independently from oxygen,nitrogen or sulfur; wherein the phenylene and heteroarylene groups areunsubstituted or substituted with 1 to 4 substituents selectedindependently from halo, (1-4C)alkyl or (1-4C)alkoxy; wherein each alkyland alkoxy group is optionally substituted with from 1 to 3 fluorosubstituents;

n is 0, 1, 2, 3 or 4;

provided that the values of m, n and Ar¹ are selected such that thenumber of contiguous atoms in the chain —(CH₂)_(m)—Ar¹—(CH₂)_(n)—between the two nitrogen atoms to which it is attached is in the rangeof from 7 to 12;

R⁵ is selected from hydrogen, (1-6C)alkyl, Ar², —CH₂Ar² and—CH₂CH₂NHC(O)R^(5a); where Ar² represents phenyl, (3-6C)cycloalkyl or(3-5C)heteroaryl containing 1 or 2 heteroatoms selected from oxygen,nitrogen or sulfur, wherein the phenyl and heteroaryl groups areunsubstituted or substituted with 1 to 3 substituents selectedindependently from halo, (1-4C)alkyl, (1-4C)alkoxy and methylenedioxy;and wherein R^(5a) represents (1-4C)alkyl;

R⁶ is hydrogen or (1-6C)alkyl; or R⁵ and R⁶ together with the nitrogenatom to which they are attached form a (3-5C)azacycloalkyl group; orwhen Ar¹ represents heteroarylene, R⁵ and R⁶ together with the nitrogenatom to which they are attached can additionally form a morpholin-1-ylor 4-(1-6C)alkylpiperazin-1-yl group; and

wherein each alkyl group in R¹, R², R³, R^(4a), R^(4b), R⁵, R⁶ andR^(a-c) is optionally substituted with from 1 to 5 fluoro substituents;

or a pharmaceutically acceptable salt or solvate or stereoisomerthereof.

In another of its composition aspects, this invention is directed to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of a compound of formulaI or a pharmaceutically acceptable salt or solvate or stereoisomerthereof. Such pharmaceutical compositions may optionally contain othertherapeutic agents. Accordingly, in one embodiment, this invention isdirected to such a pharmaceutical composition wherein the compositionfurther comprises a therapeutically effective amount of a steroidalanti-inflammatory agent, such as a corticosteroid; a β₂ adrenergicreceptor agonist; a phosphodiesterase-4 inhibitor; or a combinationthereof.

Compounds of this invention possess muscarinic receptor antagonistactivity. Accordingly, compounds of formula I are expected to be usefulfor treating pulmonary disorders, such as chronic obstructive pulmonarydisease and asthma.

Accordingly, in one of its method aspects, this invention is directed toa method for treating a pulmonary disorder, the method comprisingadministering to a patient a therapeutically effective amount of acompound of formula I or a pharmaceutically acceptable salt or solvateor stereoisomer thereof.

Additionally, in another of its method aspects, this invention isdirected to a method of producing bronchodilation in a patient, themethod comprising administering to a patient a bronchodilation-producingamount of a compound of formula I or a pharmaceutically acceptable saltor solvate or stereoisomer thereof.

This invention is also directed to a method of treating chronicobstructive pulmonary disease or asthma, the method comprisingadministering to a patient a therapeutically effective amount of acompound of formula I or a pharmaceutically acceptable salt or solvateor stereoisomer thereof.

In another one of its method aspects, this invention is directed to amethod for antagonizing a muscarinic receptor in a mammal comprisingadministering to the mammal, a therapeutically effective amount of thecompound of formula I.

Since compounds of this invention possess muscarinic receptor antagonistactivity, such compounds are also useful as research tools. Accordingly,in yet another of its method aspects, this invention is directed to amethod for using a compound of formula I or a pharmaceuticallyacceptable salt or solvate or stereoisomer thereof as a research toolfor studying a biological system or sample, or for discovering newchemical compounds having muscarinic receptor antagonist activity.

This invention is also directed to processes and novel intermediatesuseful for preparing compounds of formula I or a pharmaceuticallyacceptable salt or solvate or stereoisomer thereof. Accordingly, inanother of its method aspects, this invention is directed to a processof preparing a compound of formula I, the process comprising:

-   -   (a) reacting a compound of formula II with a compound of formula        III;    -   (b) reacting a compound of formula IV with a compound of formula        V in the presence of a reducing agent;    -   (c) reacting a compound of formula VI with a compound of formula        V;    -   (d) reacting a compound of formula II with a compound of formula        VII in the presence of a reducing agent; or    -   (e) reacting a compound of formula VIII with a reducing agent;        to provide a compound of formula I; wherein compounds of formula        II-VIII are as defined herein.

In one embodiment, the above process further comprises the step offorming a pharmaceutically acceptable salt of a compound of formula I.In other embodiments, this invention is directed to the other processesdescribed herein; and to the product prepared by any of the processesdescribed herein.

This invention is also directed to a compound of formula I or apharmaceutically acceptable salt or solvate or stereoisomer thereof, foruse in therapy or as a medicament.

Additionally, this invention is directed to the use of a compound offormula I or a pharmaceutically acceptable salt or solvate orstereoisomer thereof, for the manufacture of a medicament; especiallyfor the manufacture of a medicament for the treatment of a pulmonarydisorder or for antagonizing a muscarinic receptor in a mammal.

DETAILED DESCRIPTION OF THE INVENTION

In one of its composition aspects, this invention is directed to noveldiphenylmethyl compounds of formula I or pharmaceutically acceptablesalts or solvates or stereoisomers thereof. These compounds may containone or more chiral centers and therefore, this invention is directed toracemic mixtures; pure stereoisomers (i.e., enantiomers ordiastereomers); stereoisomer-enriched mixtures and the like unlessotherwise indicated. When a particular stereoisomer is shown or namedherein, it will be understood by those skilled in the art that minoramounts of other stereoisomers may be present in the compositions ofthis invention unless otherwise indicated, provided that the desiredutility of the composition as a whole is not eliminated by the presenceof such other isomers.

In particular, when e is 1, the compounds of formula I contain a chiralcenter at the carbon atom indicated by the symbol * in the followingpartial formula (shown without optional substituents for clarity):

In one embodiment of this invention, the carbon atom identified by thesymbol * has the (R) configuration. In this embodiment, it is preferredfor compounds of formula I to have the (R) configuration at the carbonatom identified by the symbol * or to be enriched in a stereoisomericform having the (R) configuration at this carbon atom. In anotherembodiment of this invention, the carbon atom identified by the symbol *has the (S) configuration. In this embodiment, it is preferred forcompounds of formula I to have the (S) configuration at the carbon atomidentified by the symbol * or to be enriched in a stereoisomeric formhaving the (S) configuration at this carbon atom.

The compounds of formula I also contain several basic groups (e.g.,amino groups) and therefore, the compounds of formula I can exist as thefree base or in various salt forms. All such salt forms are includedwithin the scope of this invention. Furthermore, solvates of compoundsof formula I or salts thereof are included within the scope of thisinvention.

Additionally, where applicable, all cis-trans or E/Z isomers (geometricisomers), tautomeric forms and topoisomeric forms of the compounds offormula I are included within the scope of this invention unlessotherwise specified.

The compounds of formula I, as well as those compounds used in itssynthesis, may also include isotopically-labeled compounds, i.e., whereone or more atoms have been enriched with atoms having an atomic massdifferent from the atomic mass predominately found in nature. Examplesof isotopes that may be incorporated into the compounds of Formula (I)include, but are not limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O and ¹⁷O.

The nomenclature used herein to name the compounds of this invention isillustrated in the Examples herein. This nomenclature has generally beenderived using the commercially-available AutoNom software (MDL, SanLeandro, Calif.).

Representative Embodiments

The following substituents and values are intended to providerepresentative examples of various aspects and embodiments of thisinvention. These representative values are intended to further defineand illustrate such aspects and embodiments and are not intended toexclude other embodiments or to limit the scope of this invention. Inthis regard, the representation that a particular value or substituentis preferred is not intended in any way to exclude other values orsubstituents from this invention unless specifically indicated.

The values for a and b are independently 0, 1, 2, 3, 4 or 5;particularly independently 0, 1 or 2; and even more particularly 0 or 1.In one embodiment, both a and b are 0.

When present, each R¹ and R² may be at the 2, 3, 4, 5 or 6-position ofthe phenyl ring to which it is attached. Each R¹ and R² areindependently selected from (1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl,(3-6C)cycloalkyl, cyano, halo, —OR^(a), —SR^(a), —NR^(a)R^(b),—S(O)R^(c) and —S(O)₂R^(c). Each R^(a) and R^(b) independentlyrepresents hydrogen, (1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl or(3-6C)cycloalkyl. Each R^(c) independently represents (1-4C)alkyl,(2-4C)alkenyl, (2-4C)alkynyl or (3-6C)cycloalkyl. Alternatively, twoadjacent R¹ groups or two adjacent R² groups may be joined together toform (3-6C)alkylene, (2-4C)alkylene-O— or —O-(2-4C)alkylene-O—. Inaddition, each alkyl group in R¹, R², and R^(a-c) is optionallysubstituted with from 1 to 5 fluoro substituents, and in one embodimentoptionally substituted with 1 to 3 fluoro substituents. In a specificembodiment, R¹ or R² are independently selected from (1-4C)alkyl,fluoro, chloro and —OR^(a). In another specific embodiment, each R¹ andR² is (1-2C)alkyl or fluoro. Representative R¹ and R² groups include,but are not limited to, methyl, ethyl, n-propyl, isopropyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, fluoro, chloro,methoxy, ethoxy, difluoromethoxy and trifluoromethoxy.

The value for c is 0, 1, 2, or 3; particularly 0, 1 or 2; and even moreparticularly 0 or 1. A particular value for c is 0. Particular mentionis made of compounds in which each of a, b and c represents 0.

When present, each R³ independently is fluoro or (1-4C)alkyl. Inaddition, each alkyl group in R³ is optionally substituted with from 1to 5 fluoro substituents, and in one embodiment optionally substitutedwith 1 to 3 fluoro substituents. In a specific embodiment, each R³ isindependently selected from (1-2C)alkyl and fluoro. When two R³substituents are present (c=2), they can be on the same or differentcarbon atoms. Representative R³ groups include, but are not limited to,methyl, ethyl, difluoromethyl, trifluoromethyl and fluoro.

R^(4a) and R^(4b) are independently selected from hydrogen, (1-4C)alkyl,and phenyl-(1-4C)alkyl. R^(4a) and R^(4b) together with the nitrogenatom to which they are attached may form a (3-6C)heterocyclic ringoptionally containing one additional heteroatom selected from nitrogen,oxygen or sulfur. This heterocyclic ring is unsubstituted or substitutedwith 1 or 2 substituents selected independently from (1-4C)alkyl andfluoro. In addition, each alkyl group in R^(4a) and R^(4b) is optionallysubstituted with from 1 to 5 fluoro substituents, and in one embodimentoptionally substituted with 1 to 3 fluoro substituents. In oneembodiment, R^(4a) and R^(4b) are independently hydrogen or (1-4C)alkyl.In another embodiment R^(4a) and R^(4b) are independently hydrogen or(1-2C)alkyl, such as methyl and ethyl. In yet another embodiment, R^(4a)and R^(4b) are both hydrogen. Alternatively, in another specificembodiment, R^(4a) and R^(4b) are joined together with the nitrogen atomto which they are attached to form a (3-5C)heterocyclic ring optionallycontaining one additional heteroatom selected from nitrogen, oxygen orsulfur. Representative heterocyclic rings include, but are not limitedto, pyrrolidin-1-yl, piperidin-1-yl, piperazin-1-yl,4-(1-4C)alkylpiperazin-1-yl, morpholin-4-yl and thiomorpholin-4-yl.

The value for e is 1 or 2. In one embodiment, e is 1.

The value for m is 1, 2, 3 or 4. In one embodiment, m is 2, 3 or 4. Inanother embodiment, m is 2 or 3.

Ar¹ represents a phenylene group or a (3-5C)heteroarylene groupcontaining 1 or 2 heteroatoms selected independently from oxygen,nitrogen or sulfur. The phenylene and heteroarylene groups areunsubstituted or substituted with 1 to 4 substituents selectedindependently from halo, (1-4C)alkyl or (1-4C)alkoxy; where each alkyland alkoxy group is optionally substituted with from 1 to 3 fluorosubstituents;

In one embodiment Ar¹ is phen-1,3-ylene or phen-1,4-ylene wherein thephenylene group is unsubstituted or substituted with 1, 2 or 3substituents selected independently from halo, (1-4C)alkyl or(1-4C)alkoxy, wherein each alkyl and alkoxy group is optionallysubstituted with from 1 to 3 fluoro substituents. Representativesubstituents include fluoro, chloro, methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, methoxy, ethoxy, isopropoxy,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl andtrifluoromethoxy. Particular examples of Ar¹ groups in this embodimentinclude 2-fluorophen-1,4-ylene, 3-fluorophen-1,4-ylene,2-chlorophen-1,4-ylene, 3-chlorophen-1,4-ylene, 2-methylphen-1,4-ylene,3-methylphen-1,4-ylene, 2-methoxyphen-1,4-ylene,3-methoxyphen-1,4-ylene, 2-trifluoromethoxyphen-1,4-ylene,3-trifluoromethoxyphen-1,4-ylene, 2,3-difluorophen-1,4-ylene,2,5-difluorophen-1,4-ylene, 2,6-difluorophen-1,4-ylene,2,3-dichlorophen-1,4-ylene, 2,5-dichlorophen-1,4-ylene,2,6-dichlorophen-1,4-ylene, 2-chloro-5-methoxyphen-1,4-ylene,2-chloro-6-methoxyphen-1,4-ylene,2-chloro-5-trifluoromethoxyphen-1,4-ylene and2-chloro-6-trifluoromethoxyphen-1,4-ylene.

In another embodiment, Ar¹ is a (3-5C)heteroarylene group containing 1or 2 heteroatoms selected independently from oxygen, nitrogen or sulfur;wherein the heteroarylene group is unsubstituted or substituted with 1or 2 substituents selected independently from halo, (1-4C)alkyl or(1-4C)alkoxy; wherein each alkyl and alkoxy group is optionallysubstituted with from 1 to 3 fluoro substituents. Representativeheteroarylene groups include divalent species of pyrrole, imidazole,thiazole, oxazole, furan, thiophene, pyrazole, isoxazole, isothiazole,pyridine, pyrazine, pyridazine and pyrimidine, where the point ofattachment is at any available carbon or nitrogen ring atom. Morespecific examples of such Ar¹ groups include 2,5-furylene,2,4-thienylene, 2,5-thienylene, 2,5-pyridylene, 2,6-pyridylene, and2,5-pyrrolylene. Representative R⁵ substituents include fluoro, chloro,methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, methoxy, ethoxy, isopropoxy, difluoromethyl,trifluoromethyl, 2,2,2-trifluoroethyl and trifluoromethoxy. Particularexamples of substituted Ar¹ groups include 3-fluoro-2,5-thienylene,3-chloro-2,5-thienylene, 3-methyl-2,5-thienylene,3-methoxy-2,5-thienylene, and 3-methoxy-6-chloro-2,5-pyridylene.

The value for n is 0, 1, 2, 3 or 4. In one embodiment, n is 0, 2 or 3.In another embodiment, n is 2 or 3.

The values of m, n and Ar¹ are selected such that the number ofcontiguous atoms in the chain —(CH₂)_(m)—Ar¹—(CH₂)_(n)— between the twonitrogen atoms to which it is attached is in the range of from 7 to 12;

R⁵ is selected from hydrogen, (1-6C)alkyl, Ar², —CH₂Ar² and—CH₂CH₂NHC(O)R^(5a). Ar² represents phenyl, (3-6C)cycloalkyl or(3-5C)heteroaryl containing 1 or 2 heteroatoms selected from oxygen,nitrogen or sulfur, where the phenyl and heteroaryl groups areunsubstituted or substituted with 1 to 3 substituents selectedindependently from halo, (1-4C)alkyl, (1-4C)alkoxy and methylenedioxy.R^(5a) represents (1-4C)alkyl. In addition, each alkyl group in R⁵ isoptionally substituted with from 1 to 5 fluoro substituents, and in oneembodiment optionally substituted with 1 to 3 fluoro substituents. In aspecific embodiment, R⁵ is hydrogen or (1-4C)alkyl, for examplehydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl and tert-butyl. In another embodiment, R⁵ is Ar², where Ar² is(3-6C)cycloalkyl, such as cyclopropyl, cyclobutyl and cyclopentyl. Inyet another embodiment, R⁵ is —CH₂Ar², where Ar² is a phenyl, furyl,thienyl, pyridyl or pyrazinyl group that is optionally substituted asdescribed above, for example, 3,4-methylenedioxyphenylmethyl,fur-2-ylmethyl and 5-methylpyrazin-2-ylmethyl. In still anotherembodiment, R⁵ is —CH₂CH₂NHCOR^(5a), such as —CH₂CH₂NHCOCH₃. An exampleof a particular value for R^(5a) is methyl.

R⁶ is hydrogen or (1-6C)alkyl, including hydrogen, methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. Inaddition, each alkyl group in R⁶ is optionally substituted with from 1to 5 fluoro substituents, and in one embodiment optionally substitutedwith 1 to 3 fluoro substituents. In a specific embodiment, R⁶ ishydrogen or methyl.

Alternatively, R⁵ and R⁶ together with the nitrogen atom to which theyare attached form a (3-5C)azacycloalkyl group, examples of whichinclude, azetidin-1-yl, pyrrolidin-1-yl and piperidin-1-yl groups. Inaddition, when Ar¹ represents heteroarylene, such as thienylene, R⁵ andR⁶ together with the nitrogen atom to which they are attached can form amorpholin-1-yl or 4-(1-6C)alkylpiperazin-1-yl group.

A particular group of compounds of interest are compounds of formula Iwherein a and b are 0. Another group of compounds of interest arecompounds of formula I wherein a, b and c are 0. A particular group ofcompounds of interest are compounds of formula I wherein a, b and c are0; and R^(4a) and R^(4b) are hydrogen. Another particular group ofcompounds of interest are compounds of formula I wherein a, b and c are0; R^(4a) and R^(4b) are hydrogen; and R⁵ is (1-4C)alkyl. Anotherparticular group of compounds of interest are compounds of formula Iwherein a, b and c are 0; R^(4a) and R^(4b) are hydrogen; R⁵ is(1-4C)alkyl; and R⁶ is hydrogen or methyl. Another particular group ofcompounds of interest are compounds of formula I wherein a, b and c are0; R^(4a) and R^(4b) are hydrogen; and R⁵ and R⁶ are joined togetherwith nitrogen to which they are attached to form a (3-5C)azacycloalkylgroup.

Another particular group of compounds of interest are compounds offormula I, where a, b, and c are 0; R^(4a) and R^(4b) are hydrogen; e is1; and Ar¹ is phen-1,4-ylene. These compounds have the formula Ia:

where m, n, R⁵ and R⁶ are as defined herein; where m+n is an integerfrom 3 to 8; and where the phen-1,4-ylene group is optionallysubstituted with 1 to 4 substituents as defined herein for Ar¹; or apharmaceutically acceptable salt or solvate or stereoisomer thereof.

Another particular group of compounds of interest are compounds offormula I, where a, b, and c are 0; R^(4a) and R^(4b) are hydrogen; e is1; and Ar¹ is phen-1,3-ylene. These compounds have the formula Ib:

where m, n, R⁵ and R⁶ are as defined herein; where m+n is an integerfrom 4 to 9; and where the phen-1,3-ylene group is optionallysubstituted with 1 to 4 substituents as defined herein for Ar¹; or apharmaceutically acceptable salt or solvate or stereoisomer thereof.

Another particular group of compounds of interest are compounds offormula I, where a, b, and c are 0; R^(4a) and R^(4b) are hydrogen; e is1; and Ar¹ is 2,5-thiophene. These compounds have the formula Ic:

where m, n, R⁵ and R⁶ are as defined herein; where m+n is an integerfrom 4 to 9; and where the 2,5-thienylene group is optionallysubstituted with 1 to 2 substituents as defined herein for Ar¹; or apharmaceutically acceptable salt or solvate or stereoisomer thereof.

Another particular group of compounds of interest are compounds offormula I, where a, b, and c are 0; R^(4a) and R^(4b) are hydrogen; ande is 1. These compounds have the formula Id:

where Ar¹, R⁵, R⁶, m and n are as defined in Table I; or apharmaceutically acceptable salt or solvate thereof.

TABLE I Ex. m Ar¹ n R⁵ R⁶ 1 4 phen-1,4-ylene 0 H H 2 2 phen-1,4-ylene 2—CH₃ H 3 2 phen-1,3-ylene 2 —CH₃ H 4 3 2,5-thienylene 3 —CH₃ H 5 3phen-1,3-ylene 3 —CH₃ H 6 3 phen-1,4-ylene 2 —CH₃ H 7 3 2,5-thienylene 3—CH₂CH₃ H 8 3 2,5-thienylene 3 —CH(CH₃)₂ H 9 3 2,5-thienylene 3 —CH₃—CH₃ 10 2 phen-1,4-ylene 2 —CH₂CH₃ H 11 2 phen-1,4-ylene 2 —CH(CH₃)₂ H12 2 phen-1,4-ylene 2 —CH₃ —CH₃ 13 2 phen-1,4-ylene 2 —(CH₂)₄—* 14 2phen-1,4-ylene 2 —CH₂-phenyl —CH₃ 15 2 phen-1,4-ylene 2 —CH₂CH₂NHC(O)CH₃H 16 2 phen-1,4-ylene 2 —CH₂-(5-methylpyrazin-2-yl) H 17 2phen-1,4-ylene 2 —CH₂-(furan-2-yl) H 18 2 phen-1,4-ylene 2—CH₂-(3,4-methylenedioxy- H phenyl) 19 2 phen-1,4-ylene 2 —(CH₂)₃—* 20 32,5-thienylene 3 —CH₂CH₂NHC(O)CH₃ H 21 3 2,5-thienylene 3—CH₂-(5-methylpyrazin-2-yl) H 22 3 2,5-thienylene 3 —CH₂-(furan-2-yl) H23 3 2,5-thienylene 3 —CH₂-(3,4-methylenedioxy- H phenyl) 24 32,5-thienylene 3 —(CH₂CH₂N(CH₃)CH₂CH₂—* 25 3 2,5-thienylene 3—(CH₂CH₂OCH₂CH₂—* 26 3 2,5-thienylene 3 —(CH₂)₃—* *In these compounds,R⁵ and R⁶ are joined together to form the group indicated.Definitions

When describing the compounds, compositions, methods and processes ofthis invention, the following terms have the following meanings unlessotherwise indicated.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to 10 carbon atoms. Representative alkyl groupsinclude, by way of example, methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl,n-nonyl, n-decyl and the like.

The term “alkylene” means a divalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from 1 to 10 carbon atoms. Representativealkylene groups include, by way of example, methylene, ethane-1,2-diyl(“ethylene”), propane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl,pentane-1,5-diyl and the like.

The term “alkoxy” means a monovalent group of the formula (alkyl)-O—,where alkyl is as defined herein. Representative alkoxy groups include,by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,sec-butoxy, isobutoxy, tert-butoxy and the like.

The term “alkenyl” means a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon double bonds. Unless otherwisedefined, such alkenyl groups typically contain from 2 to 10 carbonatoms. Representative alkenyl groups include, by way of example,ethenyl, n-propenyl, isopropenyl, n-but-2-enyl, n-hex-3-enyl and thelike. The term “alkenylene” means a divalent alkenyl group.

The term “alkynyl” means a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon triple bonds. Unless otherwisedefined, such alkynyl groups typically contain from 2 to 10 carbonatoms. Representative alkynyl groups include, by way of example,ethynyl, n-propynyl, n-but-2-ynyl, n-hex-3-ynyl and the like. The term“alkynylene” means a divalent alkynyl group.

The term “aryl” means a monovalent aromatic hydrocarbon having a singlering (i.e., phenyl) or fused rings (i.e., naphthalene). Unless otherwisedefined, such aryl groups typically contain from 6 to 10 carbon ringatoms. Representative aryl groups include, by way of example, phenyl andnaphthalene-1-yl, naphthalene-2-yl, and the like. The term “arylene”means a divalent aryl group.

The term “azacycloalkyl” means a monovalent heterocyclic ring containingone nitrogen atom, i.e., a cycloalkyl group in which one carbon atom hasbeen replaced with a nitrogen atom. Unless otherwise defined, suchazacycloalkyl groups typically contain from 2 to 9 carbon atoms.Representative examples of an azacycloalkyl group are pyrrolidinyl andpiperidinyl groups. The term “azacycloalkylene” means a divalentazacycloakyl group. Representative examples of an azacycloalkylene groupare pyrrolidinylene and piperidinylene groups.

The term “cycloalkyl” means a monovalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkyl groupstypically contain from 3 to 10 carbon atoms. Representative cycloalkylgroups include, by way of example, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl and the like. The term “cycloalkylene” means a divalentcycloalkyl group.

The term “halo” means fluoro, chloro, bromo and iodo.

The term “heteroaryl” means a monovalent aromatic group having a singlering or two fused rings and containing in the ring at least oneheteroatom (typically 1 to 3 heteroatoms) selected from nitrogen, oxygenor sulfur. Unless otherwise defined, such heteroaryl groups typicallycontain from 5 to 10 total ring atoms. Representative heteroaryl groupsinclude, by way of example, monovalent species of pyrrole, imidazole,thiazole, oxazole, furan, thiophene, triazole, pyrazole, isoxazole,isothiazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine,indole, benzofuran, benzothiophene, benzimidazole, benzthiazole,quinoline, isoquinoline, quinazoline, quinoxaline and the like, wherethe point of attachment is at any available carbon or nitrogen ringatom. The term “heteroarylene” means a divalent heteroaryl group.

The term “heterocyclyl” or “heterocyclic” means a monovalent saturatedor unsaturated (non-aromatic) group having a single ring or multiplecondensed rings and containing in the ring at least one heteroatom(typically 1 to 3 heteroatoms) selected from nitrogen, oxygen or sulfur.Unless otherwise defined, such heterocyclic groups typically containfrom 2 to 9 total ring carbon atoms. Representative heterocyclic groupsinclude, by way of example, monovalent species of pyrrolidine,imidazolidine, pyrazolidine, piperidine, 1,4-dioxane, morpholine,thiomorpholine, piperazine, 3-pyrroline and the like, where the point ofattachment is at any available carbon or nitrogen ring atom. The term“heterocyclene” means a divalent heterocyclyl or heterocyclic group.

When a specific number of carbon atoms is intended for a particular termused herein, the number of carbon atoms is shown in parenthesespreceding the term. For example, the term “(1-4C)alkyl” means an alkylgroup having from 1 to 4 carbon atoms.

The term “pharmaceutically acceptable salt” means a salt which isacceptable for administration to a patient, such as a mammal (e.g.,salts having acceptable mammalian safety for a given dosage regime).Such salts can be derived from pharmaceutically acceptable inorganic ororganic bases and from pharmaceutically acceptable inorganic or organicacids. Salts derived from pharmaceutically acceptable inorganic basesinclude ammonium, calcium, copper, ferric, ferrous, lithium, magnesium,manganic, manganous, potassium, sodium, zinc and the like. Particularlypreferred are ammonium, calcium, magnesium, potassium and sodium salts.Salts derived from pharmaceutically acceptable organic bases includesalts of primary, secondary and tertiary amines, including substitutedamines, cyclic amines, naturally-occurring amines and the like, such asarginine, betaine, caffeine, choline, N,N′-dibenzylethylenediamine,diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol,ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine,glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. Salts derived frompharmaceutically acceptable acids include acetic, ascorbic,benzenesulfonic, benzoic, camphosulfonic, citric, ethanesulfonic,edisylic, fumaric, gentisic, gluconic, glucoronic, glutamic, hippuric,hydrobromic, hydrochloric, isethionic, lactic, lactobionic, maleic,malic, mandelic, methanesulfonic, mucic, naphthalenesulfonic,naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic, nicotinic,nitric, orotic, pamoic, pantothenic, phosphoric, succinic, sulfuric,tartaric, p-toluenesulfonic, xinafoic and the like. Particularlypreferred are citric, hydrobromic, hydrochloric, isethionic, maleic,naphthalene-1,5-disulfonic, phosphoric, sulfuric and tartaric acids.

The term “salt thereof” means a compound formed when the hydrogen of anacid is replaced by a cation, such as a metal cation or an organiccation and the like. Preferably, the salt is a pharmaceuticallyacceptable salt, although this is not required for salts of intermediatecompounds that are not intended for administration to a patient.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, i.e. a compound of formula I or apharmaceutically acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. Representativesolvents include, by way of example, water, methanol, ethanol,isopropanol, acetic acid and the like. When the solvent is water, thesolvate formed is a hydrate.

It will be appreciated that the term “or a pharmaceutically acceptablesalt or solvate or stereoisomer thereof” is intended to include allpermutations of salts, solvates and stereoisomers, such as a solvate ofa pharmaceutically acceptable salt of a stereoisomer of a compound offormula I.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as COPD) in a patient,such as a mammal (particularly a human) that includes:

-   -   (a) preventing the disease or medical condition from occurring,        i.e., prophylactic treatment of a patient;    -   (b) ameliorating the disease or medical condition, i.e.,        eliminating or causing regression of the disease or medical        condition in a patient;    -   (c) suppressing the disease or medical condition, i.e., slowing        or arresting the development of the disease or medical condition        in a patient; or    -   (d) alleviating the symptoms of the disease or medical condition        in a patient.

The term “leaving group” means a functional group or atom which can bedisplaced by another functional group or atom in a substitutionreaction, such as a nucleophilic substitution reaction. By way ofexample, representative leaving groups include chloro, bromo and iodogroups; sulfonic ester groups, such as mesylate, tosylate, brosylate,nosylate and the like; and acyloxy groups, such as acetoxy,trifluoroacetoxy and the like.

The term “protected derivatives thereof” means a derivative of thespecified compound in which one or more functional groups of thecompound are protected from undesired reactions with a protecting orblocking group. Functional groups which may be protected include, by wayof example, carboxylic acid groups, amino groups, hydroxyl groups, thiolgroups, carbonyl groups and the like. Representative protecting groupsfor carboxylic acids include esters (such as a p-methoxybenzyl ester),amides and hydrazides; for amino groups, carbamates (such astert-butoxycarbonyl) and amides; for hydroxyl groups, ethers and esters;for thiol groups, thioethers and thioesters; for carbonyl groups,acetals and ketals; and the like. Such protecting groups are well-knownto those skilled in the art and are described, for example, in T. W.Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, ThirdEdition, Wiley, N.Y., 1999, and references cited therein.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino group. Representativeamino-protecting groups include, but are not limited to,tert-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl (Cbz),9-fluorenylmethoxycarbonyl (Fmoc), formyl, trimethylsilyl (TMS),tert-butyldimethylsilyl (TBS), and the like.

The term “carboxy-protecting group” means a protecting group suitablefor preventing undesired reactions at a carboxy group. Representativecarboxy-protecting groups include, but are not limited to, esters, suchas methyl, ethyl, tert-butyl, benzyl (Bn), p-methoxybenzyl (PMB),9-fluroenylmethyl (Fm), trimethylsilyl (TMS), tert-butyldimethylsilyl(TBS), diphenylmethyl (benzhydryl, DPM) and the like.

The term “hydroxyl-protecting group” means a protecting group suitablefor preventing undesirable reactions at a hydroxyl group. Representativehydroxyl-protecting groups include, but are not limited to, silyl groupsincluding tri(1-6C)alkylsilyl groups, such as trimethylsilyl (TMS),triethylsilyl (TES), tert-butyldimethylsilyl (TBS) and the like; esters(acyl groups) including (1-6C)alkanoyl groups, such as formyl, acetyland the like; arylmethyl groups, such as benzyl (Bn), p-methoxybenzyl(PMB), 9-fluorenylmethyl (Fm), diphenylmethyl (benzhydryl, DPM) and thelike. Additionally, two hydroxyl groups can also be protected as analkylidene group, such as prop-2-ylidine, formed, for example, byreaction with a ketone, such as acetone.

General Synthetic Procedures

The diphenylmethyl compounds of this invention can be prepared fromreadily available starting materials using the following general methodsand procedures or by using other information readily available to thoseof ordinary skill in the art. Although a particular embodiment of thepresent invention may be shown or described herein, those skilled in theart will recognize that all embodiments or aspects of the presentinvention can be prepared using the methods described herein or by usingother methods, reagents and starting materials known to those skilled inthe art. It will also be appreciated that where typical or preferredprocess conditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. While the optimumreaction conditions may vary depending on the particular reactants orsolvent used, such conditions can be readily determined by one skilledin the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Thechoice of a suitable protecting group for a particular functional groupas well as suitable conditions for protection and deprotection of suchfunctional groups are well-known in the art. Protecting groups otherthan those illustrated in the procedures described herein may be used,if desired. For example, numerous protecting groups, and theirintroduction and removal, are described in T. W. Greene and G. M. Wuts,Protecting Groups in Organic Synthesis, Third Edition, Wiley, N.Y.,1999, and references cited therein.

By way of illustration, the compounds of formula I can be prepared by aprocess comprising:

-   -   (a) reacting a compound of formula II:

with a compound of formula III:X¹—(CH₂)_(m)—Ar¹—(CH₂)_(n)—NR⁵R⁶  IIIwherein X¹ represents a leaving group;

-   -   (b) reacting a compound of formula IV:

with a compound of formula V:HNR⁵R⁶  Vin the presence of a reducing agent;

-   -   (c) reacting a compound of formula VI:

wherein X² represents a leaving group, with a compound of formula V;

-   -   (d) reacting a compound of formula II with a compound of formula        VII:        OHC—(CH₂)_(m-1)—Ar¹—(CH₂)_(n)—NR⁵R⁶  VII        in the presence of a reducing agent; or    -   (e) reacting a compound of formula VIII:

with a reducing agent; to provide a compound of formula I; andoptionally, forming a pharmaceutically acceptable salt or solvate orstereoisomer thereof.

In these reactions, depending upon the particular substituents present,one or more protecting groups may be employed. If such protecting groupsare used, they are removed using conventional procedures to provide thecompound of formula I.

Generally, if a salt of one of the starting materials is used in theprocesses described above, such as an acid addition salt, the salt istypically neutralized before or during the reaction process. Thisneutralization reaction is typically accomplished by contacting the saltwith one molar equivalent of a base for each molar equivalent of acidaddition salt.

In process (a), the reaction between the compounds of formula II andIII, the leaving group represented by X¹ can be, for example, halo, suchas chloro, bromo or iodo, or a sulfonic ester group, such as mesylate ortosylate. The reaction is conveniently performed in the presence of abase, for example, a tertiary amine such as diisopropylethylamine.Convenient solvents include nitrites, such as acetonitrile. The reactionis conveniently conducted at a temperature in the range of from 0° C. to100° C.

Compounds of formula II may be prepared as described in U.S. Pat. No.5,096,890 to Cross et al., the disclosure of which is incorporatedherein by reference in its entirety. Alternatively, compounds of formulaII can be prepared by deprotecting a compound of formula IX:

in which P¹ represents an amino-protecting group, such as a benzylgroup. Benzyl groups are conveniently removed by reduction, for example,using a hydrogen or ammonium formate and a Group VIII metal catalyst,such as palladium.

Compounds of formula IX can be prepared by reacting a carboxylic acid offormula X:

with an amine of formula HNR^(4a)R^(4b) under amide bond formingconditions.

Compounds of formula X may be prepared by hydrolyzing a compound offormula XI:

Compounds of formula XI can be prepared as described in U.S. Pat. No.5,096,890 to Cross et al.

Compounds of formula III are generally known or can be prepared fromreadily available starting materials using well-known synthetic methods.

In process (b), the reducing agent can be, for example, hydrogen in thepresence of a Group VIII metal catalyst, such as palladium, or a metalhydride reducing agent, such as sodium triacetoxyborohydride. Thereaction is conveniently performed at a temperature in the range of from0° C. to 100° C. Convenient solvents include halogenated hydrocarbons,such as dichloroethane and alcohols, such as methanol.

Compounds of formula IV can be prepared by oxidizing a compound offormula XII:

using a suitable oxidizing agent, such as sulfur trioxide pyridinecomplex in dimethyl sulfoxide in the presence of a tertiary amine base,such as diisopropylethylamine.

Compounds of formula XII can be prepared by reacting a compound offormula II with a compound of formula XIII:X³—(CH₂)_(m)—Ar¹—(CH₂)_(n)—OH  XIIIin which X³ represents a leaving group including, for example, halo,such as chloro, bromo or iodo, or a sulfonic ester group, such asmesylate or tosylate.

In process (c), the reaction between the compounds of formula VI and V,the leaving group represented by X² can be, for example, halo, such aschloro, bromo or iodo, or a sulfonic ester group, such as mesylate ortosylate. The reaction is conveniently performed in the presence of abase such as a tertiary amine, including diisopropylethylamine, or analkali metal carbonate, such as potassium carbonate. Convenient solventsinclude nitrites, such as acetonitrile and amides, such asdimethylformamide. The reaction is conveniently conducted at atemperature in the range of from 0° C. to 100° C.

Compounds of formula VI can be prepared by reacting a compound offormula XII with an appropriate reagent, for example, a halogenatingagent such as thionyl chloride. Alternatively, such compounds can beprepared by reacting a compound of formula II with a compound of formulaXIV:X²—(CH₂)_(m)—Ar¹—(CH₂)_(n)—X²  XIV

The compounds of formula XIV may conveniently be prepared by reactingthe corresponding diol with a halogenating agent. For example, compoundsof formula XIV in which X² represents bromine may be prepared byreacting the corresponding diol with a brominating agent, such ashydrogen bromide or dibromotriphenylphosporane.

In process (d), the reducing agent can be, for example, hydrogen in thepresence of a Group VIII metal catalyst, such as palladium, or a metalhydride reducing agent, such as sodium triacetoxyborohydride. Thereaction is conveniently performed at a temperature in the range of from0° C. to 100° C. Convenient solvents include halogenated hydrocarbons,such as dichloroethane and alcohols, such as methanol. Compounds offormula VII can be prepared by oxidizing a compound of formula XV:HO—(CH₂)_(m)—Ar¹—(CH₂)_(n)—NR⁵R⁶  XVusing a suitable oxidizing agent, such as sulfur trioxide pyridinecomplex in dimethyl sulfoxide in the presence of a tertiary amine base,such as diisopropylethylamine.

In process (e), the reducing agent can be, for example, a metal hydridereducing agent, such as diisobutylaluminium hydride (DIBAL). Thereaction is conveniently performed at a temperature in the range of from0° C. to 100° C. Suitable solvents include ethers, such astetrahydrofuran. Compounds of formula VIII can be prepared by reacting acompound of formula II with a compound of formula XVI:HOOC—(CH₂)_(m-1)—Ar¹—(CH₂)_(n)—NR⁵R⁶  XVIor a reactive derivative thereof, such as an anhydride. The reaction maybe conducted under conventional amide bond-forming conditions.

It will be appreciated that certain compounds of formula I, for examplethose in which R⁵ represents —(CH₂)Ar², such as benzyl, may themselvesserve as intermediates for other compounds of formula I. Thus, a—(CH₂)Ar² group may serve as a protecting group and may be removed bycatalytic hydrogenation, for example, in the presence of palladium oncarbon.

Certain of the intermediates described herein are believed to be noveland accordingly, such compounds are provided as further aspects of theinvention including, for example, the compounds of formula IV, VI andVIII and salts thereof.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of this invention orintermediates thereof are described in the Examples set forth below.

Pharmaceutical Compositions and Formulations

The diphenylmethyl compounds of this invention are typicallyadministered to a patient in the form of a pharmaceutical composition orformulation. Such pharmaceutical compositions may be administered to thepatient by any acceptable route of administration including, but notlimited to, inhaled, oral, nasal, topical (including transdermal) andparenteral modes of administration. It will be understood that any formof the compounds of this invention, (i.e., free base, pharmaceuticallyacceptable salt, solvate, etc.) that is suitable for the particular modeof administration can be used in the pharmaceutical compositionsdiscussed herein.

Accordingly, in one of its composition aspects, this invention isdirected to a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier or excipient and a therapeutically effective amountof a compound of formula I, or a pharmaceutically acceptable salt orsolvate or stereoisomer thereof. Optionally, such pharmaceuticalcompositions may contain other therapeutic and/or formulating agents ifdesired.

The pharmaceutical compositions of this invention typically contain atherapeutically effective amount of a compound of the present inventionor a pharmaceutically acceptable salt or solvate or stereoisomerthereof. Typically, such pharmaceutical compositions will contain fromabout 0.01 to about 95% by weight of the active agent; including, fromabout 0.01 to about 30% by weight; such as from about 0.01 to about 10%by weight of the active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of this invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the ingredients for such compositionsare commercially available from, for example, Sigma, P.O. Box 14508, St.Louis, Mo. 63178. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following: (1)sugars, such as lactose, glucose and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;(4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)excipients, such as cocoa butter and suppository waxes; (9) oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; (10) glycols, such as propylene glycol; (11)polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;(12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions;(21) compressed propellant gases, such as chlorofluorocarbons andhydrofluorocarbons; and (22) other non-toxic compatible substancesemployed in pharmaceutical compositions.

The pharmaceutical compositions of this invention are typically preparedby thoroughly and intimately mixing or blending a compound of theinvention with a pharmaceutically acceptable carrier and one or moreoptional ingredients. If necessary or desired, the resulting uniformlyblended mixture can then be shaped or loaded into tablets, capsules,pills, canisters, cartridges, dispensers and the like using conventionalprocedures and equipment.

In one embodiment, the pharmaceutical compositions of this invention aresuitable for inhaled administration. Suitable pharmaceuticalcompositions for inhaled administration will typically be in the form ofan aerosol or a powder. Such compositions are generally administeredusing well-known delivery devices, such as a nebulizer inhaler, ametered-dose inhaler (MDI), a dry powder inhaler (DPI) or a similardelivery device.

In a specific embodiment of this invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a nebulizer inhaler. Such nebulizer devices typically produce astream of high velocity air that causes the pharmaceutical compositioncomprising the active agent to spray as a mist that is carried into thepatient's respiratory tract. Accordingly, when formulated for use in anebulizer inhaler, the active agent is typically dissolved in a suitablecarrier to form a solution. Alternatively, the active agent can bemicronized and combined with a suitable carrier to form a suspension ofmicronized particles of respirable size, where micronized is typicallydefined as having about 90% or more of the particles with a diameter ofless than about 10 μm. Suitable nebulizer devices are providedcommercially, for example, by PARI GmbH (Stamberg, German). Othernebulizer devices include Respimat (Boehringer Ingelheim) and thosedisclosed, for example, in U.S. Pat. No. 6,123,068 to Lloyd et al. andWO 97/12687 (Eicher et al.).

A representative pharmaceutical composition for use in a nebulizerinhaler comprises an isotonic aqueous solution comprising from about0.05 μg/mL to about 10 mg/mL of a compound of formula I or apharmaceutically acceptable salt or solvate or stereoisomer thereof.

In another specific embodiment of this invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a dry powder inhaler. Such dry powder inhalers typicallyadminister the active agent as a free-flowing powder that is dispersedin a patient's air-stream during inspiration. In order to achieve a freeflowing powder, the active agent is typically formulated with a suitableexcipient such as lactose or starch.

A representative pharmaceutical composition for use in a dry powderinhaler comprises dry lactose having a particle size between about 1 μmand about 100 μm and micronized particles of a compound of formula I, ora pharmaceutically acceptable salt or solvate or stereoisomer thereof.

Such a dry powder formulation can be made, for example, by combining thelactose with the active agent and then dry blending the components.Alternatively, if desired, the active agent can be formulated without anexcipient. The pharmaceutical composition is then typically loaded intoa dry powder dispenser, or into inhalation cartridges or capsules foruse with a dry powder delivery device.

Examples of dry powder inhaler delivery devices include Diskhaler(GlaxoSmithKline, Research Triangle Park, N.C.) (see, e.g., U.S. Pat.No. 5,035,237 to Newell et al.); Diskus (GlaxoSmithKline) (see, e.g.,U.S. Pat. No. 6,378,519 to Davies et al.); Turbuhaler (AstraZeneca,Wilmington, Del.) (see, e.g., U.S. Pat. No. 4,524,769 to Wetterlin);Rotahaler (GlaxoSmithKline) (see, e.g., U.S. Pat. No. 4,353,365 toHallworth et al.) and Handihaler (Boehringer Ingelheim). Furtherexamples of suitable DPI devices are described in U.S. Pat. No.5,415,162 to Casper et al., U.S. Pat. No. 5,239,993 to Evans, and U.S.Pat. No. 5,715,810 to Armstrong et al., and references cited therein.

In yet another specific embodiment of this invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a metered-dose inhaler. Such metered-dose inhalers typicallydischarge a measured amount of the active agent or a pharmaceuticallyacceptable salt or solvate or stereoisomer thereof using compressedpropellant gas. Accordingly, pharmaceutical compositions administeredusing a metered-dose inhaler typically comprise a solution or suspensionof the active agent in a liquefied propellant. Any suitable liquefiedpropellant may be employed including chlorofluorocarbons, such as CCl₃F,and hydrofluoroalkanes (HFAs), such as 1,1,1,2-tetrafluoroethane (HFA134a) and 1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227). Due toconcerns about chlorofluorocarbons affecting the ozone layer,formulations containing HFAs are generally preferred. Additionaloptional components of HFA formulations include co-solvents, such asethanol or pentane, and surfactants, such as sorbitan trioleate, oleicacid, lecithin, and glycerin. See, for example, U.S. Pat. No. 5,225,183to Purewal et al., EP 0717987 A2 (Minnesota Mining and ManufacturingCompany), and WO 92/22286 (Minnesota Mining and Manufacturing Company).

A representative pharmaceutical composition for use in a metered-doseinhaler comprises from about 0.01% to about 5% by weight of a compoundof formula I, or a pharmaceutically acceptable salt or solvate orstereoisomer thereof; from about 0% to about 20% by weight ethanol; andfrom about 0% to about 5% by weight surfactant; with the remainder beingan HFA propellant.

Such compositions are typically prepared by adding chilled orpressurized hydrofluoroalkane to a suitable container containing theactive agent, ethanol (if present) and the surfactant (if present). Toprepare a suspension, the active agent is micronized and then combinedwith the propellant. The formulation is then loaded into an aerosolcanister, which forms a portion of a metered-dose inhaler device.Examples of metered-dose inhaler devices developed specifically for usewith HFA propellants are provided in. U.S. Pat. No. 6,006,745 to Mareckiand U.S. Pat. No. 6,143,277 to Ashurst et al. Alternatively, asuspension formulation can be prepared by spray drying a coating ofsurfactant on micronized particles of the active agent. See, forexample, WO 99/53901 (Glaxo Group Ltd.) and WO 00/61108 (Glaxo GroupLtd.).

For additional examples of processes of preparing respirable particles,and formulations and devices suitable for inhalation dosing see U.S.Pat. Nos. 6,268,533 to Gao et al., U.S. Pat. No. 5,983,956 to Trofast,U.S. Pat. No. 5,874,063 to Briggner et al., and U.S. Pat. No. 6,221,398to Jakupovic et al.; and WO 99/55319 (Glaxo Group Ltd.) and WO 00/30614(AstraZeneca AB).

In another embodiment, the pharmaceutical compositions of this inventionare suitable for oral administration. Suitable pharmaceuticalcompositions for oral administration may be in the form of capsules,tablets, pills, lozenges, cachets, dragees, powders, granules; or as asolution or a suspension in an aqueous or non-aqueous liquid; or as anoil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup;and the like; each containing a predetermined amount of a compound ofthe present invention as an active ingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof this invention will typically comprise a compound of the presentinvention as the active ingredient and one or more pharmaceuticallyacceptable carriers, such as sodium citrate or dicalcium phosphate.Optionally or alternatively, such solid dosage forms may also comprise:(1) fillers or extenders, such as starches, lactose, sucrose, glucose,mannitol, and/or silicic acid; (2) binders, such ascarboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as cetyl alcohol and/or glycerol monostearate; (8)absorbents, such as kaolin and/or bentonite clay; (9) lubricants, suchas talc, calcium stearate, magnesium stearate, solid polyethyleneglycols, sodium lauryl sulfate, and/or mixtures thereof; (10) coloringagents; and (11) buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of this invention. Examples ofpharmaceutically acceptable antioxidants include: (1) water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal-chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like. Coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate (CAP), polyvinyl acetatephthalate (PVAP), hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate(CAT), carbokymethyl ethyl cellulose (CMEC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), and the like.

If desired, the pharmaceutical compositions of the present invention mayalso be formulated to provide slow or controlled release of the activeingredient using, by way of example, hydroxypropyl methyl cellulose invarying proportions; or other polymer matrices, liposomes and/ormicrospheres.

In addition, the pharmaceutical compositions of the present inventionmay optionally contain opacifying agents and may be formulated so thatthey release the active ingredient only, or preferentially, in a certainportion of the gastrointestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Such liquid dosage formstypically comprise the active ingredient and an inert diluent, such as,for example, water or other solvents, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (e.g., cottonseed, groundnut, corn, germ,olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminium metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionsof this invention are preferably packaged in a unit dosage form. Theterm “unit dosage form” means a physically discrete unit suitable fordosing a patient, i.e., each unit containing a predetermined quantity ofactive agent calculated to produce the desired therapeutic effect eitheralone or in combination with one or more additional units. For example,such unit dosage forms may be capsules, tablets, pills, and the like.

The compounds of this invention can also be administered transdermallyusing known transdermal delivery systems and excipients. For example, acompound of this invention can be admixed with permeation enhancers,such as propylene glycol, polyethylene glycol monolaurate,azacycloalkan-2-ones and the like, and incorporated into a patch orsimilar delivery system. Additional excipients including gelling agents,emulsifiers and buffers, may be used in such transdermal compositions ifdesired.

The pharmaceutical compositions of this invention may also contain othertherapeutic agents that are co-administered with a compound of formulaI, or pharmaceutically acceptable salt or solvate or stereoisomerthereof. For example, the pharmaceutical compositions of this inventionmay further comprise one or more therapeutic agents selected from otherbronchodilators (e.g., PDE₃ inhibitors, adenosine 2b modulators and β₂adrenergic receptor agonists); anti-inflammatory agents (e.g., steroidalanti-inflammatory agents, such as corticosteroids; non-steroidalanti-inflammatory agents (NSAIDs), and PDE₄ inhibitors); othermuscarinic receptor antagonists (i.e., antichlolinergic agents);antiinfective agents (e.g., Gram positive and Gram negative antibioticsor antivirals); antihistamines; protease inhibitors; and afferentblockers (e.g., D₂ agonists and neurokinin modulators). In oneparticular aspect of the invention, the compound of the invention isco-administered with a β₂ adrenergic receptor agonist and a steroidalanti-inflammatory agent. The other therapeutic agents can be used in theform of pharmaceutically acceptable salts or solvates. Additionally, ifappropriate, the other therapeutic agents can be used as optically purestereoisomers.

Representative β₂ adrenergic receptor agonists that can be used incombination with the compounds of this invention include, but are notlimited to, salmeterol, salbutamol, formoterol, salmefamol, fenoterol,terbutaline, albuterol, isoetharine, metaproterenol, bitolterol,pirbuterol, levalbuterol and the like, or pharmaceutically acceptablesalts thereof. Other β₂ adrenergic receptor agonists that can be used incombination with the compounds of this invention include, but are notlimited to,3-(4-{[6-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-phenyl]ethyl}amino)-hexyl]oxy}butyl)benzenesulfonamideand3-(-3-{[7-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}-amino)heptyl]oxy}-propyl)benzenesulfonamideand related compounds disclosed in WO 02/066422 (Glaxo Group Ltd.);3-[3-(4-{[6-([(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino)hexyl]oxy}butyl)-phenyl]imidazolidine-2,4-dioneand related compounds disclosed in WO 02/070490 (Glaxo Group Ltd.);3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)-hexyl]oxy}butyl)-benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamide and related compounds disclosed in WO02/076933 (Glaxo Group Ltd.);4-{(1R)-2-[(6-{2-[(2,6-dichlorobenzyl)oxy]ethoxy}hexyl)amino]-1-hydroxyethyl}-2-(hydroxymethyl)phenoland related compounds disclosed in WO 03/024439 (Glaxo Group Ltd.);N-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamineand related compounds disclosed in U.S. Pat. No. 6,576,793 to Moran etal.;N-{2-[4-(3-phenyl-4-methoxyphenyl)aminophenyl]ethyl}-(R)-2-hydroxy-2-(8-hydroxy-2(1H)-quinolinon-5-yl)ethylamineand related compounds disclosed in U.S. Pat. No. 6,653,323 to Moran etal.; and pharmaceutically acceptable salts thereof. In a particularembodiment, the β₂-adrenoreceptor agonist is a crystallinemonohydrochloride salt ofN-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamine.When employed, the β₂-adrenoreceptor agonist will be present in thepharmaceutical composition in a therapeutically effective amount.Typically, the β₂-adrenoreceptor agonist will be present in an amountsufficient to provide from about 0.05 μg to about 500 μg per dose.

Representative steroidal anti-inflammatory agents that can be used incombination with the compounds of this invention include, but are notlimited to, methyl prednisolone, prednisolone, dexamethasone,fluticasone propionate,6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester,6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-androsta-1,4-diene-17β-carbothioicacid S-(2-oxo-tetrahydrofuran-3S-yl) ester, beclomethasone esters (e.g.,the 17-propionate ester or the 17,21-dipropionate ester), budesonide,flunisolide, mometasone esters (e.g., the furoate ester), triamcinoloneacetonide, rofleponide, ciclesonide, butixocort propionate, RPR-106541,ST-126 and the like, or pharmaceutically-acceptable salts thereof. Whenemployed, the steroidal anti-inflammatory agent will be present in thepharmaceutical composition in a therapeutically effective amount.Typically, the steroidal anti-inflammatory agent will be present in anamount sufficient to provide from about 0.05 μg to about 500 μg perdose.

An exemplary combination is a compound of formula I, or pharmaceuticallyacceptable salt or solvate or stereoisomer thereof, co-administered withsalmeterol as the β₂ adrenergic receptor agonist, and fluticasonepropionate as the steroidal anti-inflammatory agent. Another exemplarycombination is a compound of formula I, or pharmaceutically acceptablesalt or solvate or stereoisomer thereof, co-administered with acrystalline monohydrochloride salt ofN-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamineas the β₂-adrenoreceptor agonist, and6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester as the steroidal anti-inflammatory agent.

Other suitable combinations include, for example, otheranti-inflammatory agents, e.g., NSAIDs (such as sodium cromoglycate;nedocromil sodium; phosphodiesterase (PDE) inhibitors (e.g.,theophylline, PDE4 inhibitors or mixed PDE3/PDE4 inhibitors);leukotriene antagonists (e.g., monteleukast); inhibitors of leukotrienesynthesis; iNOS inhibitors; protease inhibitors, such as tryptase andelastase inhibitors; beta-2 integrin antagonists and adenosine receptoragonists or antagonists (e.g., adenosine 2 a agonists); cytokineantagonists (e.g., chemokine antagonists such as, an interleukinantibody (αIL antibody), specifically, an αIL-4 therapy, an αIL-13therapy, or a combination thereof); or inhibitors of cytokine synthesis.

For example, representative phosphodiesterase-4 (PDE4) inhibitors ormixed PDE3/PDE4 inhibitors that can be used in combination with thecompounds of this invention include, but are not limited to cis4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylicacid,2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-one;cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol];cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylicacid and the like, or pharmaceutically acceptable salts thereof. Otherrepresentative PDE4 or mixed PDE4/PDE3 inhibitors include AWD-12-281(elbion); NCS-613 (INSERM); D-4418 (Chiroscience and Schering-Plough);CI-1018 or PD-168787 (Pfizer); benzodioxole compounds disclosed inWO99/16766 (Kyowa Hakko); K-34 (Kyowa Hakko); V-11294A (Napp);roflumilast (Byk-Gulden); pthalazinone compounds disclosed in WO99/47505(Byk-Gulden); Pumafentrine (Byk-Gulden, now Altana); arofylline(Almirall-Prodesfarma); VM554/UM565 (Vemalis); T-440 (Tanabe Seiyaku);and T2585 (Tanabe Seiyaku).

Representative muscarinic antagonists (i.e., anticholinergic agents)that can be used in combination with, and in addition to, the compoundsof this invention include, but are not limited to, atropine, atropinesulfate, atropine oxide, methylatropine nitrate, homatropinehydrobromide, hyoscyamine (d, l) hydrobromide, scopolamine hydrobromide,ipratropium bromide, oxitropium bromide, tiotropium bromide,methantheline, propantheline bromide, anisotropine methyl bromide,clidinium bromide, copyrrolate (Robinul), isopropamide iodide,mepenzolate bromide, tridihexethyl chloride (Pathilone), hexocycliummethylsulfate, cyclopentolate hydrochloride, tropicamide,trihexyphenidyl hydrochloride, pirenzepine, telenzepine, AF-DX 116 andmethoctramine and the like, or a pharmaceutically acceptable saltthereof; or, for those compounds listed as a salt, alternatepharmaceutically acceptable salt thereof.

Representative antihistamines (i.e., H₁-receptor antagonists) that canbe used in combination with the compounds of this invention include, butare not limited to, ethanolamines, such as carbinoxamine maleate,clemastine fumarate, diphenylhydramine hydrochloride and dimenhydrinate;ethylenediamines, such as pyrilamine amleate, tripelennaminehydrochloride and tripelennamine citrate; alkylamines, such aschlorpheniramine and acrivastine; piperazines, such as hydroxyzinehydrochloride, hydroxyzine pamoate, cyclizine hydrochloride, cyclizinelactate, meclizine hydrochloride and cetirizine hydrochloride;piperidines, such as astemizole, levocabastine hydrochloride, loratadineor its descarboethoxy analogue, terfenadine and fexofenadinehydrochloride; azelastine hydrochloride; and the like, or apharmaceutically acceptable salt thereof; or, for those compounds listedas a salt, alternate pharmaceutically acceptable salt thereof.

Suitable doses for the other therapeutic agents administered incombination with a compound of the invention are in the range of about0.05 μg/day to about 100 mg/day.

The following formulations illustrate representative pharmaceuticalcompositions of the present invention:

Formulation Example A

A dry powder for administration by inhalation is prepared as follows:

Ingredients Amount Compound of the invention 0.2 mg Lactose  25 mg

Representative Procedure: The compound of the invention is micronizedand then blended with lactose. This blended mixture is then loaded intoa gelatin inhalation cartridge. The contents of the cartridge areadministered using a powder inhaler.

Formulation Example B

A dry powder formulation for use in a dry powder inhalation device isprepared as follows:

Representative Procedure: A pharmaceutical composition is preparedhaving a bulk formulation ratio of micronized compound of the inventionto lactose of 1:200. The composition is packed into a dry powderinhalation device capable of delivering between about 10 μg and about100 μg of the compound of the invention per dose.

Formulation Example C

A dry powder for administration by inhalation in a metered dose inhaleris prepared as follows:

Representative Procedure: A suspension containing 5 wt % of a compoundof the invention and 0.1 wt % lecithin is prepared by dispersing 10 g ofthe compound of the invention as micronized particles with mean sizeless than 10 μm in a solution formed from 0.2 g of lecithin dissolved in200 mL of demineralized water. The suspension is spray dried and theresulting material is micronized to particles having a mean diameterless than 1.5 μm. The particles are loaded into cartridges withpressurized 1,1,1,2-tetrafluoroethane.

Formulation Example D

A pharmaceutical composition for use in a metered dose inhaler isprepared as follows:

Representative Procedure: A suspension containing 5 wt % compound of theinvention, 0.5 wt % lecithin, and 0.5 wt % trehalose is prepared bydispersing 5 g of active ingredient as micronized particles with meansize less than 10 μm in a colloidal solution formed from 0.5 g oftrehalose and 0.5 g of lecithin dissolved in 100 mL of demineralizedwater. The suspension is spray dried and the resulting material ismicronized to particles having a mean diameter less than 1.5 μm. Theparticles are loaded into canisters with pressurized1,1,1,2-tetrafluoroethane.

Formulation Example E

A pharmaceutical composition for use in a nebulizer inhaler is preparedas follows:

Representative Procedure: An aqueous aerosol formulation for use in anebulizer is prepared by dissolving 0.1 mg of the compound of theinvention in 1 mL of a 0.9% NaCl solution acidified with citric acid.The mixture is stirred and sonicated until the active ingredient isdissolved. The pH of the solution is adjusted to a value in the range offrom 3 to 8 by the slow addition of NaOH.

Formulation Example F

Hard gelatin capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 250 mg Lactose(spray-dried) 200 mg Magnesium stearate  10 mg

Representative Procedure: The ingredients are thoroughly blended andthen loaded into a hard gelatin capsule (460 mg of composition percapsule).

Formulation Example G

A suspension for oral administration is prepared as follows:

Ingredients Amount Compound of the invention 1.0 g Fumaric acid 0.5 gSodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 gGranulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum k(Vanderbilt Co.) 1.0 g Flavoring 0.035 mL Colorings 0.5 mg Distilledwater q.s. to 100 mL

Representative Procedure: The ingredients are mixed to form a suspensioncontaining 100 mg of active ingredient per 10 mL of suspension.

Formulation Example H

An injectable formulation is prepared as follows:

Ingredients Amount Compound of the invention 0.2 g Sodium acetate buffersolution (0.4 M) 2.0 mL HCl (0.5 N) or NaOH (0.5 N) q.s. to pH 4 Water(distilled, sterile) q.s. to 20 mL

Representative Procedure: The above ingredients are blended and the pHis adjusted to 4±0.5 using 0.5 N HCl or 0.5 N NaOH.

Utility

The diphenylmethyl compounds of this invention are expected to be usefulas muscarinic receptor antagonists and therefore, such compounds areexpected to be useful for treating medical conditions mediated bymuscarinic receptors, i.e., medical conditions which are ameliorated bytreatment with a muscarinic receptor antagonist. Such medical conditionsinclude, by way of example, pulmonary disorders or diseases includingthose associated with reversible airway obstruction, such as chronicobstructive pulmonary disease (e.g., chronic and wheezy bronchitis andemphysema), asthma, pulmonary fibrosis, allergic rhinitis, rhinorrhea,and the like. Other medical conditions that can be treated withmuscarinic receptor antagonists are genitourinary tract disorders, suchas overactive bladder or detrusor hyperactivity and their symptoms;gastrointestinal tract disorders, such as irritable bowel syndrome,diverticular disease, achalasia, gastrointestinal hypermotilitydisorders and diarrhea; cardiac arrhythmias, such as sinus bradycardia;Parkinson's disease; cognitive disorders, such as Alzheimer's disease;dismenorrhea; and the like.

In one embodiment, the compounds of this invention are useful fortreating smooth muscle disorders in mammals, including humans and theircompanion animals (e.g., dogs, cats etc.). Such smooth muscle disordersinclude, by way of illustration, overactive bladder, chronic obstructivepulmonary disease and irritable bowel syndrome.

When used to treat smooth muscle disorders or other conditions mediatedby muscarinic receptors, the compounds of this invention will typicallybe administered orally, rectally, parenterally or by inhalation in asingle daily dose or in multiple doses per day. The amount of activeagent administered per dose or the total amount administered per daywill typically be determined by the patient's physician and will dependon such factors as the nature and severity of the patients condition,the condition being treated, the age and general health of the patient,the tolerance of the patient to the active agent, the route ofadministration and the like.

Typically, suitable doses for treating smooth muscle disorders or otherdisorders mediated by muscarinic receptors will range from about 0.14μg/kg/day to about 7 mg/kg/day of active agent; including from about0.15 μg/kg/day to about 5 mg/kg/day. For an average 70 kg human, thiswould amount to about 10 μg per day to about 500 mg per day of activeagent.

In a specific embodiment, the compounds of this invention are useful fortreating pulmonary or respiratory disorders, such as COPD or asthma, inmammals including humans. When used to treat such disorders, thecompounds of this invention will typically be administered by inhalationin multiple doses per day, in a single daily dose or a single weeklydose. Generally, the dose for treating a pulmonary disorder will rangefrom about 10 μg/day to about 200 μg/day. As used herein, COPD includeschronic obstructive bronchitis and emphysema (see, for example, Barnes,Chronic Obstructive Pulmonary Disease, N Engl J Med 343:269-78 (2000)).

When used to treat a pulmonary disorder, the compounds of this inventionare optionally administered in combination with other therapeutic agentssuch as a β₂-adrenoreceptor agonist; a corticosteroid, a non-steroidalanti-inflammatory agent, or combinations thereof.

When administered by inhalation, the compounds of this inventiontypically have the effect of producing bronchodilation. Accordingly, inanother of its method aspects, this invention is directed to a method ofproducing bronchodilation in a patient, the method comprisingadministering to a patient a bronchodilation-producing amount of acompound of the invention. Generally, the therapeutically effective dosefor producing bronchodilation will range from about 10 μg/day to about200 μg/day.

In another embodiment, the compounds of this invention are used to treatoveractive bladder. When used to treat overactive bladder, the compoundsof this invention will typically be administered orally in a singledaily dose or in multiple doses per day; preferably in a single dailydose. Preferably, the dose for treating overactive bladder will rangefrom about 1.0 to about 500 mg/day.

In yet another embodiment, the compounds of this invention are used totreat irritable bowel syndrome. When used to treat irritable bowelsyndrome, the compounds of this invention will typically be administeredorally or rectally in a single daily dose or in multiple doses per day.Preferably, the dose for treating irritable bowel syndrome will rangefrom about 1.0 to about 500 mg/day.

Since compounds of this invention are muscarinic receptor antagonists,such compounds are also useful as research tools for investigating orstudying biological systems or samples having muscarinic receptors. Suchbiological systems or samples may comprise M₁, M₂, M₃, M₄ and/or M₅muscarinic receptors. Any suitable biological system or sample havingmuscarinic receptors may be employed in such studies which may beconducted either in vitro or in vivo. Representative biological systemsor samples suitable for such studies include, but are not limited to,cells, cellular extracts, plasma membranes, tissue samples, mammals(such as mice, rats, guinea pigs, rabbits, dogs, pigs, etc.), and thelike.

In this embodiment, a biological system or sample comprising amuscarinic receptor is contacted with a muscarinic receptor-antagonizingamount of a compound of this invention. The effects of antagonizing themuscarinic receptor are then determined using conventional proceduresand equipment, such as radioligand binding assays and functional assays.Such functional assays include ligand-mediated changes in intracellularcyclic adenosine monophosphate (cAMP), ligand-mediated changes inactivity of the enzyme adenylyl cyclase (which synthesizes cAMP),ligand-mediated changes in incorporation of guanosine5′-O-(γ-thio)triphosphate ([³⁵S]GTPγS) into isolated membranes viareceptor catalyzed exchange of [³⁵S]GTPγS for GDP, ligand-mediatedchanges in free intracellular calcium ions (measured, for example, witha fluorescence-linked imaging plate reader or FLIPR® from MolecularDevices, Inc.). A compound of this invention will antagonize or decreasethe activation of muscarinic receptors in any of the functional assayslisted above, or assays of a similar nature. A muscarinicreceptor-antagonizing amount of a compound of this invention willtypically range from about 0.1 nanomolar to about 100 nanomolar.

Additionally, the compounds of this invention can be used as researchtools for discovering new compounds that have muscarinic receptorantagonist activity. In this embodiment, muscarinic receptor bindingdata (e.g., as determined by in vitro radioligand displacement assays)for a test compound or a group of test compounds is compared to themuscarinic receptor binding data for a compound of this invention toidentify those test compounds that have about equal or superiormuscarinic receptor binding, if any. This aspect of the inventionincludes, as separate embodiments, both the generation of comparisondata (using the appropriate assays) and the analysis of the test data toidentify test compounds of interest.

In another embodiment, the compounds of this invention are used toantagonize a muscarinic receptor in biological system, and a mammal inparticular, such as mice, rats, guinea pigs, rabbits, dogs, pigs, humansand so forth. In this embodiment, a therapeutically effective amount ofthe compound of formula I is administered to the mammal. The effects ofantagonizing the muscarinic receptor can then determined usingconventional procedures and equipment, examples of which are describedabove.

Among other properties, compounds of this invention have been found tobe potent inhibitors of M₃ muscarinic receptor activity. Accordingly, ina specific embodiment, this invention is directed to compounds offormula I having an inhibition dissociation constant (K_(i)) for the M₃receptor subtype of less than or equal to 100 nM; preferably, less thanor equal to 50 nM; and more preferably, less than or equal to 10 nM (asdetermined, for example, by an in vitro radioligand displacement assay).

Additionally, compounds of this invention are expected to possess adesirable duration of action. Accordingly, in another specificembodiment, this invention is directed to compounds of formula I havinga duration of action greater than about 24 hours.

Moreover, compounds of this invention are also expected to possessreduced side effects, such as dry mouth, at efficacious doses whenadministered by inhalation compared to other known muscarinic receptorantagonists administered by inhalation (such as tiotropium).

These properties, as well as the utility of the compounds of thisinvention, can be demonstrated using various in vitro and in vivo assayswell-known to those skilled in the art. For example, representativeassays are described in further detail in the following Examples.

EXAMPLES

The following Preparations and Examples illustrate specific embodimentsof this invention. In these examples, the following abbreviations havethe following meanings:

AC adenylyl cyclase ACh acetylcholine ACN acetonitrile BSA bovine serumalbumin cAMP 3′-5′ cyclic adenosine monophosphate CHO Chinese hamsterovary cM₅ cloned chimpanzee M₅ receptor DABCO1,4-diazabicyclo[2.2.2]octane DCM dichloromethane (i.e., methylenechloride) DIBAL diisobutylaluminum hydride DIPEAN,N-diisopropylethylamine dPBS Dulbecco's phosphate buffered saline DMFdimethylformamide DMSO dimethyl sulfoxide EDTA ethylenediaminetetraacetic acid EtOAc ethyl acetate EtOH ethanol FBS fetal bovine serumFLIPR fluorometric imaging plate reader HATUO-(7-azabenzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid hM₁ cloned human M₁ receptor hM₂ cloned human M₂ receptor hM₃cloned human M₃ receptor hM₄ cloned human M₄ receptor hM₅ cloned humanM₅ receptor HPLC high-performance liquid chromatography IPAc isopropylacetate MCh methylcholine MeOH methanol MTBE methyl tert-butyl etherPyBOP benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphateTFA trifluoroacetic acid THF tetrahydrofuran

Any other abbreviations used herein but not defined have their standard,generally accepted meaning. Unless noted otherwise, all materials, suchas reagents, starting materials and solvents, were purchased fromcommercial suppliers (such as Sigma-Aldrich, Fluka, and the like) andwere used without further purification.

Unless otherwise indicated, HPLC analysis was conducted using an Agilent(Palo Alto, Calif.) Series 1100 instrument equipped with a Zorbax BonusRP 2.1×50 mm column (Agilent) having a 3.5 micron particle size.Detection was by UV absorbance at 214 nm. The mobile phases employedwere as follows (by volume): A is ACN (2%), water (98%) and TFA (0.1%);and B is ACN (90%), water (10%) and TFA (0.1%). HPLC 10-70 data wasobtained using a flow rate of 0.5 mL/minute of 10 to 70% B over a 6minute gradient (with the remainder being A). Similarly, HPLC 5-35 dataand HPLC 10-90 data were obtained using 5 to 35% B; or 10 to 90% B overa 5 minute gradient.

Liquid chromatography mass spectrometry (LCMS) data were obtained withan Applied Biosystems (Foster City, Calif.) Model API-150EX instrument.LCMS 10-90 data was obtained using 10 to 90% Mobile Phase B over a 5minute gradient.

Small-scale purification was conducted using an API 150EX PrepWorkstation system from Applied Biosystems. The mobile phases employedwere as follows (by volume): A is water and 0.05% TFA; and B is ACN and0.05% TFA. For arrays (typically about 3 to 50 mg recovered sample size)the following conditions were used: 20 mL/min flow rate; 15 mingradients and a 20 mm×50 mm Prism RP column with 5 micron particles(Thermo Hypersil-Keystone, Bellefonte, Pa.). For larger scalepurifications (typically greater than 100 mg crude sample), thefollowing conditions were used: 60 mL/min flow rate; 30 min gradientsand a 41.4 mm×250 mm Microsorb BDS column with 10 micron particles(Varian, Palo Alto, Calif.).

Example A Preparation of 2,2-Diphenyl-2-(S)-pyrrolidin-3-ylacetamide

Step A: Preparation of (S)-1-Benzyl-3-(p-toluenesulfonyloxy)pyrrolidine

To a stirred solution of (S)-1-benzyl-3-pyrrolidinol (44.3 g, 0.25 mol)and 1,4-diazabicyclo[2.2.2]octane (33.7 g, 0.3 mol) in 250 mL oftert-butyl methyl ether under an atmosphere of nitrogen at 0° C., wasadded p-toluenesulfonyl chloride (52.4 g, 0.275 mol) portion-wise over20 minutes. The reaction mixture was stirred at 0° C. for 1 hour. Theice bath was removed and the mixture was stirred at ambient temperatureovernight (20±5 h). EtOAc (100 mL) was added, followed by saturatedaqueous sodium bicarbonate solution (250 mL). The resulting mixture wasstirred at ambient temperature for 1 hour. The layers were separated andthe organic layer was washed with saturated aqueous sodium bicarbonatesolution (250 mL); saturated aqueous ammonium chloride solution (250mL); saturated aqueous NaCl solution (250 mL); and then dried oversodium sulfate (80 g). The sodium sulfate was filtered off and washedwith ethyl acetate (20 mL) and the solvent was removed in vacuo to give78.2 g of the title intermediate as an off-white solid (94% yield).

HPLC analysis of this intermediate was conducted using a YMC ODSA C184.6×50 mm column, having a 5.0 micron particle size. Detection was by UVabsorbance at 220 nm. The mobile phases employed were as follows (byvolume): A is MeOH (10%), water (90%) and TFA (0.1%); and B is MeOH(90%), water (10%) and TFA (0.1%). Using a flow rate of 4.0 mL/min of 0to 100% B in A over 5 minutes, this intermediate was determined to havea purity of 95%.

Step B: Preparation of(S)-1-Benzyl-3-(1-cyano-1,1-diphenylmethyl)-pyrrolidine

To a stirred solution of diphenylacetonitrile (12.18 g, 61.8 mmol) inanhydrous THF (120 mL) at 0° C., potassium tert-butoxide (10.60 g, 94.6mmol) was added over 5 min. The reaction mixture was stirred at 0° C.for 1 h. To the reaction mixture at 0° C. was added(S)-1-benzyl-3-(p-toluenesulfonyloxy)-pyrrolidine (20.48 g, 61.3 mmol)in one portion. The cold bath was removed and the reaction mixture wasstirred for 5-10 min at which time the reaction mixture had become abrown homogeneous solution. The reaction mixture was then heated at 40°C. overnight (20±5 h). The reaction mixture (bright yellow suspension)was allowed to cool to ambient temperature before adding water (150 mL).Most of the THF was then removed in vacuo and IPAc (200 mL) was added.The layers were separated and the organic layer was washed withsaturated aqueous ammonium chloride solution (150 mL); saturated aqueousNaCl solution (150 mL); and then dried over sodium sulfate (50 g). Thesodium sulfate was filtered off and washed with IPAc (20 mL) and thesolvent was removed in vacuo to give 23.88 g of the title intermediateas a light brown oil (>99% yield). This intermediate was determined tohave a purity of 75% (contaminated mainly with excessdiphenylacetonitriie) using the HPLC method described in Step A.

Step C: Preparation of (S)-3-(1-Cyano-1,1-diphenylmethyl)pyrrolidine

(S)-1-Benzyl-3-(1-cyano-1,1-diphenylmethyl)pyrrolidine was dissolved inIPAc (approximately 1 g/10 mL) and the solution was mixed with an equalvolume of 1N aqueous HCl. The resulting layers were separated and theaqueous layer was extracted with an equal volume of IPAc. The organiclayers were combined, dried over sodium sulfate and filtered. Thesolvent was removed in vacuo to afford(S)-1-benzyl-3-(1-cyano-1,1-diphenylmethyl)pyrrolidine hydrochloride asa light yellow foamy solid. (Note: This hydrochloride salt can also beprepared during the work-up of Step B).

To a stirred solution of(S)-1-benzyl-3-(1-cyano-1,1-diphenylmethyl)pyrrolidine hydrochloride(8.55 g, 21.98 mmol) in MeOH (44 mL) was added palladium on carbon (1.71g) and ammonium formate (6.93 g, 109.9 mmol). The reaction mixture washeated to 50° C. with stirring for 3 hours. The reaction was cooled toambient temperature and water (20 mL) was added. The resulting mixturewas filtered through a pad of Celite, washing with MeOH (20 mL). Thefiltrate was collected and most of the MeOH was removed in vacuo. Theresidue was mixed with IPAc (100 mL) and 10% aqueous sodium carbonate(50 mL). The resulting layers were separated and the aqueous layer wasextracted with IPAc (50 mL). The organic layers were combined and driedover sodium sulfate (20 g). The sodium sulfate was filtered off andwashed with IPAc (20 mL). The solvent was removed in vacuo to afford5.75 g of the title intermediate as a light yellow oil (99.7% yield, 71%purity by HPLC).

Step D: Preparation of 2,2-Diphenyl-2-(S)-pyrrolidin-3-ylacetamide

A 200 mL flask with a magnetic stir bar and a nitrogen inlet was chargedwith (S)-3-(1-cyano-1,1-diphenylmethyl)pyrrolidine (2.51 g) and 80%H₂SO₄ (19.2 mL; pre-prepared with 16 mL of 96% H₂SO₄ and 3.2 mL of H₂O).The reaction mixture was then heated at 90° C. for 24 hours or until thestarting material was consumed as indicated by HPLC. The reactionmixture was allowed to cool to ambient temperature and then poured ontoice (approximately 50 mL by volume). A 50% aqueous NaOH solution wasadded slowly to the mixture with stirring over an ice bath until the pHwas about 12. DCM (200 mL) was added and mixed with the aqueous solutionat which time sodium sulfate precipitated out and was filtered off. Thefiltrate was collected and the layers were separated. The aqueous layerwas extracted with DCM (100 mL) and the organic layers were combined anddried with over sodium sulfate (5 g). The sodium sulfate was filteredoff and washed with DCM (10 mL). The solvent was removed in vacuo togive the crude product as a light yellow foamy solid (ca. 2.2 g, 86%purity by HPLC).

The crude product was dissolved in EtOH (18 mL) with stirring. To thissolution was added a warm solution of L-tartaric acid (1.8 g) in EtOH(14 mL) and the resulting mixture was stirred overnight (15±5 h). Theresulting precipitate was isolated by filtration to give an off-whitesolid (ca. 3.2 g, >95% purity by HPLC). MeOH (15 mL) was added to thissolid and the resulting slurry was stirred at 70° C. overnight (15hours). The slurry was allowed to cool to ambient temperature and awhite solid (˜2.6 g, >99% purity by HPLC) was obtained after filtration.To this solid was added EtOAc (30 mL) and 1 N aqueous NaOH (25 mL). Thismixture was mixed until two distinct layers formed and then the layerswere separated and the aqueous layer was extracted with EtOAc (20 mL).The organic layers were combined and dried over sodium sulfate (10 g).The sodium sulfate was removed by filtration and the solvent wasevaporated in vacuo to afford 1.55 g of the title intermediate as anoff-white foamy solid (58% yield).

HPLC analysis was conducted using an Inertsil OCD-2 C18 column.Detection was by UV absorbance at 254 nm. The mobile phases employedwere as follows (by volume): A is MeOH (5%), water (95%), and TFA(0.1%); and B is MeOH (95%), water (5%) and TFA (0.1%). Using a flowrate of 1.0 mL/min of 0 to 100% B in A over 15 minutes, thisintermediate was determined to have a purity of >99%.

Example 1 Synthesis of2-{(S)-1-[4-(4-Aminophenyl)butyl]pyrrolidin-3-yl}-2,2-diphenylacetamide

Step A: Preparation of2-{(S)-1-[4-(4-Aminophenyl)butyryl]pyrrolidin-3-yl}-2,2-diphenylacetamide

Diisopropylethylamine (0.809 mL, 4.6 mmol) was added to a solution of2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide (1.00 g, 3.6 mmol) and4-(4-aminophenyl)butyric acid (0.646 g, 3.61 mmol) in DMF (17 mL) andthe resulting mixture was stirred at ambient temperature. After about 15minutes, HATU (1.63 g, 4.3 mmol) was added and the reaction mixture wasstirred for two hours. The reaction mixture was concentrated to one halfits volume and then diluted with DCM (15 mL). The layers were separatedand the organic layer was washed with saturated aqueous sodiumbicarbonate solution (3×15 mL), brine (15 mL), dried over magnesiumsulfate, and then concentrated under vacuum. The crude product waspurified on a silica gel column using MeOH (5%)/DCM (95%) as the eluentto afford 700 mg of the title intermediate as a solid. MS m/z: [M+H⁺]calc'd for C₂₈H₃₁N₃O₃ 442.2; found 442.3.

Step B: Preparation of2-{(S)-1-[4-(4-Aminophenyl)butyl]pyrrolidin-3-yl}-2,2-diphenylacetamideditrifluoroacetate salt

A solution of DIBAL in toluene (1M, 1.64 mL) was added to a solution ofthe intermediate from Step A (0.24 g, 0.54 mmol) in THF (3.6 mL) atambient temperature and the resulting mixture was stirred for 72 hours.The reaction mixture was then concentrated and the residue was added toa 1:1 mixture of water and ACN (1 mL) containing 0.01% TFA. This mixturewas then purified by HPLC to give 37.4 mg of the title compound as thebis(trifluoroacetate) salt. MS m/z: [M+H⁺] calc'd for C₂₈H₃₃N₃O 428.3;found 428.6.

Example 2 Synthesis of2-((S)-1-{2-[4-(2-Methylaminoethyl)phenyl]ethyl}pyrrolidin-3-yl)-2,2-diphenyl-acetamide

Step A: Preparation {4-[(N-Benzyl-N-methylcarbamoyl)methyl]phenyl}aceticAcid

To a 2-L three-necked flask, equipped with a magnetic stir bar, nitrogengas inlet and addition funnel, was added 1,4-phenylenediacetic acid (50g, 257 mmol), PyBOP (127.5 g, 245 mmol) and DCM (700 mL). The reactionmixture was then cooled to 0° C. to 10° C. A solution ofbenzylmethylamine (34.81 mL, 269.7 mmol) and DIPEA (85.4 mL, 490 mmol)in DCM (300 mL) was then added slowly and the reaction mixture wasstirred at ambient temperature overnight (˜16 hours). The reactionmixture was then washed with water (2×200 mL) and the organic phase wasconcentrated under reduced pressure. The pH of the resulting mixture wasadjusted with 1N NaOH to pH 10 and the mixture was then extracted withMTBE. The aqueous phase was cooled to 0° C. to 5° C. and the pH of theaqueous phase was adjusted to pH 3 by adding 1N HCl. The resultingmixture was extracted with DCM and the combined DCM layers were washedwith water (1×100 mL), brine (1×100 mL), dried over sodium sulfate andconcentrated under vacuum to afford 38 g of the title intermediate (90%purity). MS m/z: [M+H⁺] calc'd for C₁₈H₁₉NO₃ 298.1; found 298.3.

Step B: Preparation of2-{4-[2-(N-Benzyl-N-methylamino)ethyl]phenyl)ethanol

To a 500-mL three-necked flask, equipped with a magnetic stir bar,nitrogen gas inlet and addition funnel, was added the intermediate fromStep A (10 g, 33.6 mmol) and THF (200 mL). The reaction mixture wascooled to 0° C. to 10° C. and lithium aluminum hydride (118 mL, 118mmol) was added portionwise. The resulting mixture was stirred atambient temperature for 16 hours and then cooled to 0° C. to 5° C. NaOH(10M) was added slowly, followed by the addition of sodium sulfate. Thismixture was stirred for 1 hour and then filtered and concentrated. ThepH of the residue was acidified to pH 1 with 1N HCl (100 mL) and theresulting mixture was extracted with EtOAc (3×50 mL). The aqueous phasewas cooled to 0° C. to 5° C. and the pH of the aqueous phase wasadjusted to pH 14 by the addition of 50% NaOH solution. The resultingmixture was extracted with DCM (3×300 mL) and the combined organiclayers were washed with water (100 mL), brine (100 mL), dried oversodium sulfate and concentrated under vacuum to afford 7 g of the titleintermediate (7 g, 98.5% purity). MS m/z: [M+H⁺] calc'd for C₁₈H₂₃NO270.2; found 270.5.

Step C: Preparation of Toluene-4-sulfonic acid2-{4-[2-(N-benzyl-N-methylamino)ethyl]phenyl}ethyl ester

To a 1-L three-necked flask, equipped with a magnetic stir bar, anitrogen gas inlet and an addition funnel, was added the intermediatefrom Step B (22 g, 82 mmol), 1,4-diazabicyclo[2,2,2]octane (13.78 g, 123mmol) and MTBE (250 mL) and the resulting mixture was cooled to 0° C. to10° C. A solution of p-toluenesulfonyl chloride (20 g, 94.3 mmol) inMTBE (150 mL) was added slowly and the resulting mixture was stirred atambient temperature for 5 hours. The mixture was then filtered andconcentrated under reduced pressure to provide the title intermediatewhich was used in the next step without further purification. MS m/z:[M+H⁺] calc'd for C₂₅H₂₉NO₃S 424.3; found 424.3.

Step D: Preparation of2-[(S)-1-(2-{4-[2-(N-Benzyl-N-methylamino)ethyl]phenyl}ethyl)pyrrolidin-3-yl]-2,2-diphenylacetamide

To a 1-L three-necked flask, equipped with a magnetic stir bar, anitrogen gas inlet and an addition funnel, was added the intermediate ofStep C (40 g, 82 mmol), 2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide (23g, 82 mmol), DIPEA (42.8 mL, 246 mmol) and ACN (400 mL). The resultingmixture was heated at 55° C. for 12 hours and then concentrated underreduced pressure. To the residue was added DCM (1 L) was added and theresulting mixture was washed with water (1×100 mL), 20% ammoniumchloride (1×100 mL), brine (1×100 mL), dried over magnesium sulfate andconcentrated under vacuum to provide 40 g of crude material which wasfurther purified by silica gel chromatograph and reverse-phase prep-HPLCto afford 10 g of the title intermediate (98.5% purity) as thebis(trifluoroacetate) salt. MS m/z: [M+H⁺] calc'd for C₃₆H₄₁N₃O 532.3;found 532.3.

Step E: Preparation of2-((S)-1-{2-[4-(2-Methylaminoethyl)phenyl]ethyl}-pyrrolidin-3-yl)-2,2-diphenylacetamide

To a Parr hydrogenation flask was added the intermediate from Step D (6g, 11.28 mmol), palladium (60 mg, 10 wt. % (dry basis) on activatedcarbon), isopropanol (60 mL) and water (6 mL). This mixture was degassedwith nitrogen and then hydrogen was applied (˜50 psi) for 16 hrs. Themixture was filtered and the precipitate washed with isopropanol. Thefiltrate was condensed and the pH of the resulting mixture was acidifiedto pH 1 by adding 100 mL of 1N HCl. The resulting mixture was extractedwith DCM (3×50 mL) and the aqueous phase was cooled to 0° C. to 5° C.The pH of the aqueous mixture was adjusted to pH 14 by adding 50% NaOHand then the mixture was extracted with DCM (3×200 mL). The combined DCMlayers were washed with water (1×100 mL), brine (2×50 mL), dried oversodium sulfate, filtered and concentrated under vacuum to provide 4 g ofthe title compound (95% purity) as the bis(trifluoroacetate) salt. MSm/z: [M+H⁺] calc'd for C₂₉H₃₅N₃O 442.3; found 442.5.

Example 3 Synthesis of2-((S)-1-{2-[3-(2-Methylaminoethyl)phenyl]ethyl}pyrrolidin-3-yl)-2,2-diphenylacetamide

Step A: Preparation of 2-[3-(2-Hydroxyethyl)phenyl]ethanol

To a stirred solution of (3-carboxymethylphenyl)acetic acid (1.0 g, 5.15mmol) in THF (100 mL) was added slowly borane dimethyl sulfide (2.3 mL,30.9 mmol). The resulting solution was stirred overnight (˜18 hours) andthen quenched by slowly adding 1N HCl (100 mL). The mixture was thenextracted using EtOAc (3×100 mL) and the combined organic layers werewashed with saturated NaCl (100 mL), dried over magnesium sulfate,filtered and concentrated under vacuum to provide 750 mg of the titleintermediate (87% yield), which was used without further purification.

Step B: Preparation of 1,3-Bis(2-bromoethyl)benzene

To a flask was added the intermediate from Step A (500 mg, 3.01 mmol)and 48% HBr (5 mL) and the resulting mixture was heated at reflux for 6hours. The reaction mixture was then cooled to room temperature anddiluted with water (100 mL). This mixture was extracted with hexane(1×100 mL) and the organic layer was washed with water (3×100 mL),saturated NaCl (1×100 mL), dried over magnesium sulfate, filtered andconcentrated under vacuum to provide 782 mg of the title intermediate(89% yield), which was used without further purification.

Step C: Preparation of2-((S)-1-{2-[3-(2-Methylaminoethyl)phenyl]ethyl}-pyrrolidin-3-yl)-2,2-diphenylacetamide

To a flask was added 2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide (42 mg,0.15 mmol), the intermediate from Step B (44 mg, 0.15 mmol), DMF (1 mL),and potassium carbonate (62 mg, 0.45 mmol) and the resulting mixture wasstirred overnight (˜18 hours) at room temperature. Methylamine (2.0 M inTHF, 375 μL, 0.75 mmol) was then added and stirring was continued atroom temperature overnight (18 hours). The reaction mixture was thenconcentrated under vacuum and to the resulting residue was added 1:1acetic acid/water (1.0 mL). This mixture was chromatographed usingreverse-phase HPLC to obtain 19.8 mg of the title compound (20% yield)as a bis(trifluoroacetate) salt. MS m/z: [M+H⁺] calc'd for C₂₉H₃₅N₃O442.3; found 442.6. Retention time=1.90 min (10-70% ACN: H₂O, reversephase HPLC).

Example 4 Synthesis of2-((S)-1-{3-[5-(3-Methylaminopropyl)thiophen-2-yl]propyl}-pyrrolidin-3-yl)-2,2-diphenylacetamide

Step A: Preparation of 3-[5-(2-Ethoxycarbonylvinyl)thiophen-2-yl]acrylicAcid Ethyl Ester

To a stirred solution of sodium hydride (2.1 g, 53 mmol, 60% in mineraloil) in THF (200 mL) was added slowly triethyl phosphonoacetate (10 mL,50 mmol). The resulting mixture was stirred until evolution of hydrogengas ceased (˜30 minutes) and then thiophene-2,5-dicarboxaldehyde (3 g,21 mmol) was added. The resulting mixture was stirred for 1 hour andthen the solvent was removed under reduced pressure and DCM (200 mL) wasadded. The organic layer was washed with water (1×100 mL), 1N HCl (1×100mL), saturated NaCl (1×100 mL), dried over magnesium sulfate, filteredand the solvent removed under reduced pressure to provide 5.76 g of thetitle intermediate (98% yield), which was used without furtherpurification.

Step B: Preparation of3-[5-(2-Ethoxycarbonylethyl)-thiophen-2-yl]propionic Acid Ethyl Ester

A stirred solution of the intermediate from Step A (6.0 g, 21 mmol) inMeOH (200 mL) was flushed with nitrogen gas and then palladium (600 mg;10 wt. % (dry basis) on activated carbon) was added. The reaction flaskwas placed under vacuum and flushed with hydrogen gas (3 cycles). Thereaction mixture was stirred for 1 hour and then flushed with nitrogengas, filtered and the solvent removed under reduced pressure to afford6.0 g of the title intermediate (99% yield), which was used withoutfurther purification.

Step C: Preparation of 3-[5-(3-Hydroxypropyl)thiophen-2-yl]-propan-1-ol

A stirred solution of DIBAL (88 mL, 88 mmol, 1.0 M in cyclohexane) inTHF (300 mL) was cooled to −78° C. and the intermediate from Step B (5.0g, 17.6 mmol) was added dropwise. After complete addition, the reactionmixture was warmed to ambient temperature over 30 minutes. The reactionwas then quenched by slowly adding 1N HCl (200 mL) and then DCM (400 mL)was added. The organic layer was removed and the aqueous layer waswashed with DCM (4×100 mL). The combined organic layers were then washedwith saturated NaCl (100 mL), dried over magnesium sulfate, filtered andthe solvent removed under reduced pressure to provide 3.0 g of the titleintermediate (85% yield), which was used without further purification.

Step D: Preparation of 2,5-Bis(3-bromopropyl)thiophene

To a stirred solution of dibromotriphenylphosphorane (10.1 g, 24 mmol)in DCM (150 mL) was added the intermediate from Step C (1.2 g, 6.0mmol). The reaction was stirred at ambient temperatures overnight (14hours). The DCM was then removed under reduced pressure and hexane (200mL) was added to the residue. The heterogeneous mixture was stirredrapidly for 30 minutes and then the hexane was decanted from theinsoluble material. The organic layer was then washed with water (2×100mL), saturated NaCl (1×100 mL), dried over magnesium sulfate, filteredand the solvent removed under reduced pressure to provide 1.5 g of thetitle intermediate (78% yield), which was used without furtherpurification.

Step E: Preparation of2-((S)-1-{3-[5-(3-Methylaminopropyl)thiophen-2-yl]propyl}pyrrolidin-3-yl)-2,2-diphenylacetamide

To a flask was added 2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide (56 mg,0.2 mmol), the intermediate from Step D (65 mg, 0.2 mmol), DMF (2 mL)and potassium carbonate (83 mg, 0.6 mmol). The resulting mixture wasstirred overnight (18 hours) at room temperature. Methylamine (2.0 M inTHF, 0.5 mL, 1.0 mmol) was then added and the resulting mixture wasstirred at room temperature overnight (18 hours). The reaction mixturewas then concentrated under reduced pressure and a 1:1 mixture of aceticacid/water (1.0 mL) was added. This mixture was then chromatographed onreverse-phase HPLC to provide 9.7 mg of the title compound (7% yield) asa bis(trifluoroacetate) salt. MS m/z: [M+H⁺] calc'd for C₂₉H₃₇N₃OS476.3; found 476.5. Retention time=2.3 min (10-70 ACN: H₂O, reversephase HPLC).

Example 5 Synthesis of2-((S)-1-{3-[3-(3-Methylaminopropyl)phenyl]propyl}-pyrrolidin-3-yl)-2,2-diphenylacetamide

Step A: Preparation of 3-[3-(2-Ethoxycarbonylvinyl)phenyl]-acrylic AcidEthyl Ester

Triethyl phosphonoacetate (17 mL, 85.7 mmol) was slowly added to astirred solution of sodium hydride (3.7 g, 93.3 mmol, 60% in mineraloil) in THF (200 mL). The resulting mixture was stirred for about 30minutes until evolution of hydrogen gas ceased. Isophthalaldehyde (5 g,37.3 mmol) was then added and the reaction mixture was stirred for 4hours. The reaction mixture was then concentrated under reduced pressureand DCM (200 mL) was added, followed by 1N HCl (100 mL). The organiclayer was then removed and washed with water (1×100 mL), saturated NaCl(1×100 mL), dried over magnesium sulfate, filtered and the solventremoved under reduced pressure to provide 9.45 g of the titleintermediate (92% yield), which was used without further purification.

Step B: Preparation of 3-[3-(2-Ethoxycarbonylethyl)phenyl]propionic acidEthyl Ester

A stirred solution of the intermediate from Step A (6.5 g, 23.7 mmol) inMeOH (300 mL) was flushed with nitrogen gas and palladium (650 mg; 10wt. % (dry basis) on activated carbon) was added. The reaction flask wasthen placed under vacuum and flushed with hydrogen gas (3 cycles). Thereaction mixture was stirred overnight (16 hours) and then flushed withnitrogen gas. The mixture was filtered and the solvent removed underreduced pressure to provide 6.2 g of the title intermediate (94% yield),which was used without further purification.

Step C: Preparation of 3-[3-(3-Hydroxypropyl)phenyl]propan-1-ol

A stirred solution of lithium aluminum hydride (1.63 g, 42 mmol) in THF(50 mL) was cooled to 0° C. and the intermediate from Step B (6.0 g, 21mmol) in THF (50 mL) was added dropwise. After the addition wascomplete, the reaction mixture was warmed to ambient temperature over 30minutes. The reaction was quenched by slowly adding 1N HCl (200 mL) andthen DCM (400 mL) was added. The organic layer was removed and theaqueous layer was washed with DCM (4×100 mL). The combined organiclayers were washed with saturated NaCl (1×100 mL), dried over magnesiumsulfate, filtered and the solvent removed under reduced pressure toprovide 3.3 g of the title intermediate (81% yield), which was usedwithout further purification.

Step D: Preparation of 1,3-Bis(3-bromopropyl)benzene

To a stirred solution of the intermediate from Step C (2.5 g, 12.8 mmol)was added dibromotriphenylphosphorane (21.7 g, 51.5 mmol) in DCM (200mL). The resulting mixture was stirred at ambient temperature overnight(24 hours) and then the mixture was concentrated under reduced pressureand hexane (200 mL) was added to the residue. The heterogeneous mixturewas stirred rapidly for 30 minutes and then the hexane layer wasdecanted from the insoluble material. The organic layer was washed withwater (2×100 mL), saturated NaCl (1×100 mL), dried over magnesiumsulfate, filtered and the solvent removed under reduced pressure toafford 3.14 g of the title intermediate (80% yield), which was usedwithout further purification.

Step E: Preparation of2-((S)-1-{3-[3-(3-Methylaminopropyl)phenyl]propyl}-pyrrolidin-3-yl)-2,2-diphenylacetamide

To a flask was added 2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide (880mg, 1.57 mmol), the intermediate from Step D (1.0 g, 1.57 mmol), ACN (2mL) and potassium carbonate (1.4 g, 4.7 mmol) and the resulting mixturewas stirred overnight (18 hours) at ambient temperature. Methylamine(2.0 M in THF, 7.8 mL, 15.7 mmol) was added and the resulting mixturewas stirred at ambient temperature overnight (18 hours). The reactionmixture was concentrated under reduced pressure and a solution of 1:1acetic acid/water (1.0 mL) was added to the residue. This mixture waschromatographed on reverse phase silica gel (gradient elution, 10-50%ACN/water) to afford 303 mg of the title compound (27% yield) as abis(trifluoroacetate) salt. MS m/z: [M+H⁺] calc'd for C₃₁H₃₉N₃O 470.3;found 470.6. Retention time=2.61 min (10-70% ACN: H₂O, reverse phaseHPLC).

Example 6 Synthesis of2-((S)-1-{3-[4-(2-Methylaminoethyl)phenyl]propyl}-pyrrolidin-3-yl)-2,2-diphenylacetamide

Step A: Preparation of 2-[4-(2-Hydroxyethyl)phenyl]ethan-1-ol

To a stirred solution of (4-carboxymethylphenyl)acetic acid (1.0 g, 5.15mmol) in THF (100 mL) was slowly added borane dimethyl sulfide (2.3 mL,30.9 mmol) and the resulting mixture was stirred overnight (˜18 hours).The reaction was then quenched by slowly adding 1N HCl (100 mL). Theresulting mixture was then extracted with EtOAc (3×100 mL). The combinedorganic layers were washed with saturated NaCl (1×100 mL), dried overmagnesium sulfate, filtered and the solvent removed under reducedpressure to provide 750 mg of the title intermediate (87% yield), whichwas used without further purification.

Step B: Preparation of Toluene-4-sulfonic Acid2-[4-(2-Hydroxyethyl-phenyl]ethyl Ester

To a stirred solution of the intermediate from Step A in THF (50 mL) wasadded DABCO (1.51 g, 13.5 mmol) and then p-toluenesulfonyl chloride (50mL). The resulting mixture was stirred overnight at room temperature andthen filtered to remove precipitated hydrochloride salts. The filtratewas concentrated under reduced pressure and DCM (50 mL) was added to theresidue. The organic layer was then washed with water (1×100 mL),saturated NaCl (1×100 mL), dried over magnesium sulfate, filtered andthe solvent removed under reduced pressure. The resulting material waspurified by silica gel chromatography (70% EtOAc/hexane) to afford 1.25g of the title intermediate (43% yield).

Step C: Preparation of 3-[4-(2-Hydroxyethyl)phenyl]propionitrile

Sodium cyanide (1.8 g, 37 mmol) was added to a stirred solution of theintermediate from Step B (10 g, 31 mmol) in DMSO (100 mL). The mixturewas heated to 60° C. overnight (˜16 hours) and then the DMSO was removedunder reduced pressure. DCM (100 mL) was added to the residue and theresulting mixture was washed with water (1×100 mL), saturated NaCl(1×100 mL), dried over magnesium sulfate, filtered and the solventremoved under reduced pressure to provide 5.4 g of the titleintermediate (99% yield), which was used without further purification.

Step D: Preparation of3-{4-[2-(Benzylmethylamino)ethyl]phenyl}propionitrile

To a stirred solution of the intermediate from Step C (5.4 g, 30.8 mmol)in DCM (250 mL) was added DMSO (7 mL, 123.3 mmol) and DIPEA (16.1 mL,92.5 mmol). The reaction mixture was then cooled to −15° C. and sulfurtrioxide pyridine complex (14.7 g, 92.5 mmol) was added. The resultingmixture was stirred for 2 hours and then the reaction was quenched byadding 1N HCl (250 mL). This mixture was then stirred for 10 minutes andthen the organic layer was removed and washed with water (1×250 mL),saturated NaCl (1×250 mL), dried over magnesium sulfate, filtered andthe solvent removed under reduced pressure.

The residue was then immediately dissolved in DCM (200 mL) and to thissolution was added benzylmethyl amine (6.0 mL, 46.2 mmol) and sodiumtriacetoxyborohydride (13.6 g, 61.6 mmol). The reaction mixture wasstirred overnight (˜18 hours) at ambient temperature and then 1N HCl(200 mL) was added. The organic layer was removed and washed with water(1×200 mL), saturated NaCl (1×200 mL), dried over magnesium sulfate,filtered and the solvent removed under reduced pressure. The crudematerial was purified by silica gel chromatography (5% MeOH/DCM with0.6% aqueous ammonia) to afford 5.4 g of the title intermediate (52%yield).

Step E: Preparation of2-[(S)-1-(3-{4-[2-(Benzylmethylamino)-ethyl]phenyl}propyl)pyrrolidin-3-yl]-2,2-diphenylacetamide

DIBAL (22 mL, 32 mmol, 25% in toluene) was added to a stirred solutionof the intermediate from Step D (4.5 g, 16 mmol) in DCM (200 mL) at −78°C. The reaction was stirred for 3 hours and then MeOH was added until nofurther hydrogen gas evolution occurred. The mixture was stirred for 10minutes and then the organic layer was washed with 1N NaOH (1×250 mL),saturated NaCl (1×250 mL), dried over magnesium sulfate, filtered andthe solvent removed under reduced pressure.

The residue was immediately dissolved in DCM (450 mL) and to thissolution was added 2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide (6.0 g,21.3 mmol) and NaBH(OAc)₃ (6.3 g, 28.4 mmol). The resulting mixture wasstirred overnight (˜21 hours) at ambient temperature and then 1N HCl(200 mL) was added. The organic layer was removed and washed with water(1×200 mL), saturated NaCl (1×200 mL), dried over magnesium sulfate,filtered and the solvent removed under reduced pressure. The crudematerial was then purified by silica gel chromatography (5% MeOH/DCMwith 0.6% aqueous ammonia) to afford 800 mg of the title intermediate(10% yield).

Step F: Preparation of2-((S)-1-{3-[4-(2-Methylaminoethyl)phenyl]propyl}-pyrrolidin-3-yl)-2,2-diphenylacetamide

A stirred solution of the intermediate from Step E (0.8 g, 1.46 mmol) inisopropanol (100 mL) was flushed with nitrogen gas and then palladium(80 mg; 10 wt. % (dry basis) on activated carbon) was added. Thereaction flask was then placed under vacuum and then flushed withhydrogen gas (3 cycles). The reaction mixture was then stirred for 2.5hours and then was flushed with nitrogen gas, filtered and the solventremoved under reduced pressure. To the residue was added a 1:1 mixtureof acetic acid/water (1.0 mL) and this mixture was then chromatographedon reverse-phase silica gel (gradient elution, 10-50% ACN/water) toprovide 60 mg of the title compound (6% yield) as abis(trifluoroacetate) salt. MS m/z: [M+H⁺] calc'd for C₃₀H₃₇N₃O 456.3;found 456.3. Retention time=3.04 min (10-70% ACN: H₂O, reverse phaseHPLC).

Example 7 Synthesis of2-[(S)-1-{3-[5-(3-Ethylaminopropyl)thiophen-2-yl]propyl}-pyrrolidin-3-yl]-2,2-diphenylacetamide

A solution of 2,5-bis(3-bromopropyl)thiophene (32.6 mg, 0.1 mmol),2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide (28.0 mg, 0.1 mmol) andsodium bicarbonate (25.2 mg, 0.3 mmol) in DMF (0.5 mL) was stirred atroom temperature for 18 hours. Ethylamine (0.5 mmol) was added and theresulting mixture was stirred at room temperature for about 71 hours. A1:1 mixture of acetic acid/water (0.8 mL) was then added and thismixture was filtered and purified by HPLC to provide 6.6 mg of the titlecompound as a bis(trifluoroacetate) salt. MS m/z: [M+H⁺] calc'd forC₃₀H₃₉N₃OS 490.3; found 490.2.

Example 8 Synthesis of2-[(S)-1-{3-[5-(3-Isopropylaminopropyl)thiophen-2-yl]propyl}pyrrolidin-3-y]-2,2-diphenylacetamide

Using the procedure of Example 7 and substituting isopropylamine inplace of ethylamine, the title compound was prepared as abis(trifluoroacetate) salt. MS m/z: [M+H⁺] calc'd for C₃₁H₄₁N₃OS 504.3;found 504.2.

Example 9 Synthesis of2-[(S)-1-{3-[5-(3-Dimethylaminopropyl)thiophen-2-yl]-propyl}pyrrolidin-3-yl]-2,2-diphenylacetamide

Using the procedure of Example 7 and substituting dimethylamine in placeof ethylamine, the title compound was prepared as abis(trifluoroacetate) salt. MS m/z: [M+H⁺] calc'd for C₃₀H₃₉N₃OS 490.3;found 490.2.

Example 10 Synthesis of2-[(S)-1-{2-[4-(2-Ethylaminoethyl)phenyl]ethyl}pyrrolidin-3-yl]-2,2-diphenylacetamide

Step A: Preparation of 1,4-Bis(2-bromoethyl)-benzene

Dibromotriphenylphosphorane (5.07 g, 12.0 mmol) was added to a solutionof 2-[4-(2-hydroxyethyl)phenyl]ethanol (664.9 mg, 4.0 mmol) in DCM (20mL, 0.2 M) and the resulting mixture was stirred at room temperature for18 hours. The reaction mixture was concentrated under reduced pressureand hexane (100 mL) was added to the residue. This mixture was stirredat room temperature for 4 hours and then filtered. The filtrate wasconcentrated under reduced pressure to provide 1.087 g of the titleintermediate as a white solid. The precipitate was washed a second timewith hexane (100 mL) and filtered. This filtrate was concentrated underreduced pressure to afford an additional 0.053 g of the titleintermediate (combined 97% yield). ¹H NMR (CDCl₃, 300 MHz): δ 7.15-7.16(br s, 4H), 3.54 (t, J=7.7 Hz, 4H), 3.13 (t, J=7.7 Hz, 4H) ppm.

Step B: Preparation of2-[(S)-1-{2-[4-(2-ethylaminoethyl)phenyl]ethyl}-pyrrolidin-3-yl]-2,2-diphenylacetamide

A solution of 2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide (28.0 mg, 0.1mmol), 1,4-bis(2-bromoethyl)benzene (29.2 mg, 0.1 mmol) and sodiumcarbonate (41.5 mg, 0.3 mmol) in DMF (0.5 mL) was stirred at roomtemperature for 18 hours. Ethylamine (0.5 mmol) was added and theresulting mixture was stirred at room temperature for about 71 hours. A1:1 mixture of acetic acid/water (0.8 mL) was then added and thismixture was filtered and purified by RP-HPLC to provide 25.1 mg of thetitle compound as a bis(trifluoroacetate) salt. MS m/z: [M+H⁺] calc'dfor C₃₀H₃₇N₃O 456.3; found 456.2.

Using the procedures above and substituting the appropriate startingmaterials, the following compounds were prepared:

Ex. Compound MS¹ 11 2-[(S)-1-{2-[4-(2- 470.2isopropylaminoethyl)phenyl]ethyl} pyrrolidin-3-yl]-2,2-diphenylacetamide 12 2-[(S)-1-{2-[4-(2- 456.2dimethylaminoethyl)phenyl]ethyl} pyrrolidin- 3-yl]-2,2-diphenylacetamide13 2,2-diphenyl-2-[(S)-1-{2-[4-(2-pyrrolidin-1- 482.2ylethyl)phenyl]ethyl}pyrrolidin-3-yl]acetamide 142-[(S)-1-(2-{4-[2-(benzylmethylamino)ethyl]phenyl} —ethyl)pyrrolidin-3-yl]-2,2-diphenylacetamide 152-[(S)-1-(2-{4-[2-(2-acetylaminoethylamino)ethyl] 513.2phenyl}ethyl)pyrrolidin-3-yl]-2,2-diphenylacetamide 162-{(S)-1-[2-(4-{2-[(5-methylpyrazin-2-ylmethyl)amino] 534.2ethyl}phenyl)ethyl]pyrrolidin-3-yl}-2,2-diphenylacetamide 172-{(S)-1-[2-(4-{2-[(furan-2-ylmethyl)amino]ethyl} 508.2phenyl)ethyl]-pyrrolidin-3-yl}-2,2-diphenylacetamide 182-{(S)-1-[2-(4-{2-[(benzo[1,3]dioxol-5-ylmethyl)amino] 562.2ethyl}phenyl)ethyl]pyrrolidin-3-yl}-2,2-diphenylacetamide 192-((S)-1-{2-[4-(2-azetidin-1-ylethyl)phenyl]ethyl}pyrrolidin-3- 468.2yl)-2,2-diphenylacetamide 202-[(S)-1-(3-{5-[3-(2-acetylaminoethylamino)propyl]thiophen- 547.22-yl}propyl)pyrrolidin-3-yl]-2,2-diphenylacetamide 212-{(S)-1-[3-(5-{3-[(5-methylpyrazin-2-ylmethyl)amino] 568.2propyl}thiophen-2-yl)propyl]pyrrolidin-3-yl}-2,2- diphenylacetamide 222-{(S)-1-[3-(5-{3-[(furan-2-ylmethyl)amino]propyl}thiophen- 542.22-yl)-propyl]pyrrolidin-3-yl}-2,2-diphenylacetamide 232-{(S)-1-[3-(5-{3-[(benzo[1,3]dioxol-5-ylmethyl)amino] 596.2propyl}thiophen-2-yl)propyl]pyrrolidin-3-yl}-2,2- diphenylacetamide 242-[(S)-1-(3-{5-[3-(4-methylpiperazin-1-yl)propyl] thiophen-2- 545.2yl}propyl)pyrrolidin-3-yl]-2,2-diphenylacetamide 252-((S)-1-{3-[5-(3-morpholin-4-yl-propyl)thiophen-2- 532.2yl]propyl}-pyrrolidin-3-yl)-2,2-diphenylacetamide 262-((S)-1-{3-[5-(3-azetidin-1-ylpropyl)thiophen-2- 502.2yl]propyl}pyrrolidin-3-yl)-2,2-diphenylacetamide ¹Observed m/z [M + H⁺],unless otherwise indicated.

Assay 1 Radioligand Binding Assay

A. Membrane Preparation from Cells Expressing hM₁, hM₂, hM₃ and hM₄Muscarinic Receptor Subtypes

CHO cell lines stably expressing cloned human hM₁, hM₂, hM₃ and hM₄muscarinic receptor subtypes, respectively, were grown to nearconfluency in medium consisting of HAM's F-12 supplemented with 10% FBSand 250 μg/mL Geneticin. The cells were grown in a 5% CO₂, 37° C.incubator and lifted with 2 mM EDTA in dPBS. Cells were collected by 5minute centrifugation at 650×g, and cell pellets were either storedfrozen at −80° C. or membranes were prepared immediately. For membranepreparation, cell pellets were suspended in lysis buffer and homogenizedwith a Polytron PT-2100 tissue disrupter (Kinematica AG; 20 seconds×2bursts). Crude membranes were centrifuged at 40,000×g for 15 minutes at4° C. The membrane pellet was then resuspended with resuspension bufferand homogenized again with the Polytron tissue disrupter. The proteinconcentration of the membrane suspension was determined by the methoddescribed in Lowry, O. et al., Journal of Biochemistry 193:265 (1951).All membranes were stored frozen in aliquots at −80° C. or usedimmediately. Aliquots of prepared hM₅ receptor membranes were purchaseddirectly from Perkin Elmer and stored at −80° C. until use.

B. Radioligand Binding Assay on Muscarinic Receptor Subtypes hM₁, hM₂,hM₃, hM₄ and hM₅

Radioligand binding assays were performed in 96-well microtiter platesin a total assay volume of 100 μL. CHO cell membranes stably expressingeither the hM₁, hM₂, hM₃, hM₄ or hM₅ muscarinic subtype were diluted inassay buffer to the following specific target protein concentrations(μg/well): 10 μg for hM₁, 10-15 μg for hM₂, 10-20 μg for hM₃, 10-20 μgfor hM₄, and 10-12 μg for hM₅. The membranes were briefly homogenizedusing a Polytron tissue disruptor (10 seconds) prior to assay plateaddition. Saturation binding studies for determining K_(D) values of theradioligand were performed using L-[N-methyl-³H]scopolamine methylchloride ([³H]-NMS) (TRK666, 84.0 Ci/mmol, Amersham Pharmacia Biotech,Buckinghamshire, England) at concentrations ranging from 0.001 nM to 20nM. Displacement assays for determination of K_(i) values of testcompounds were performed with [³H]-NMS at 1 nM and eleven different testcompound concentrations. The test compounds were initially dissolved toa concentration of 400 μM in dilution buffer and then serially diluted5× with dilution buffer to final concentrations ranging from 10 pM to100 μM. The addition order and volumes to the assay plates were asfollows: 25 μL radioligand, 25 μL diluted test compound, and 50 μLmembranes. Assay plates were incubated for 60 minutes at 37° C. Bindingreactions were terminated by rapid filtration over GF/B glass fiberfilter plates (Perkin Elmer Inc., Wellesley, Mass.) pre-treated in 1%BSA. Filter plates were rinsed three times with wash buffer (10 mMHEPES) to remove unbound radioactivity. Plates were then air dried, and50 μL Microscint-20 liquid scintillation fluid (PerkinElmer Inc.,Wellesley, Mass.) was added to each well. The plates were then countedin a PerkinElmer Topcount liquid scintillation counter (PerkinElmerInc., Wellesley, Mass.). Binding data were analyzed by nonlinearregression analysis with the GraphPad Prism Software package (GraphPadSoftware, Inc., San Diego, Calif.) using the one-site competition model.K_(i) values for test compounds were calculated from observed IC₅₀values and the K_(D) value of the radioligand using the Cheng-Prusoffequation (Cheng Y; Prusoff W. H. Biochemical Pharmacology22(23):3099-108 (1973)). K_(i) values were converted to pK_(i) values todetermine the geometric mean and 95% confidence intervals. These summarystatistics were then converted back to K_(i) values for data reporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher binding affinity for the receptor tested. Exemplary compoundsof the invention that were tested in this assay, were found to have aK_(i) value of less than about 100 nM for the M₃ muscarinic receptorsubtype in this assay (except the compound of Example 16 which had aK_(i) of 110 nM for the M₃ muscarinic receptor). In particular, thecompounds of Examples 1 to 12 were found to have K_(i) values of lessthan 10 nM for the M₃ muscarinic receptor.

Assay 2 Muscarinic Receptor Functional Potency Assays

A. Blockade of Agonist-Mediated Inhibition of cAMP Accumulation

In this assay, the functional potency of a test compound is determinedby measuring the ability of the test compound to blockoxotremorine-inhibition of forskolin-mediated cAMP accumulation inCHO-K1 cells expressing the hM₂ receptor.

cAMP assays are performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with ¹²⁵I-cAMP (NENSMP004B, PerkinElmer Life Sciences Inc., Boston, Mass.), according tothe manufacturer's instructions.

Cells are rinsed once with dPBS and lifted with Trypsin-EDTA solution(0.05% trypsin/0.53 mM EDTA) as described in the Cell Culture andMembrane Preparation section above. The detached cells are washed twiceby centrifugation at 650×g for five minutes in 50 mLs dPBS. The cellpellet is then re-suspended in 10 mL dPBS, and the cells are countedwith a Coulter Z1 Dual Particle Counter (Beckman Coulter, Fullerton,Calif.). The cells are centrifuged again at 650×g for five minutes andre-suspended in stimulation buffer to an assay concentration of1.6×10⁶-2.8×10⁶ cells/mL.

The test compound is initially dissolved to a concentration of 400 μM indilution buffer (dPBS supplemented with 1 mg/mL BSA (0.1%)), and thenserially diluted with dilution buffer to final molar concentrationsranging from 100 μM to 0.1 nM. Oxotremorine is diluted in a similarmanner.

To measure oxotremorine inhibition of AC activity, 25 μL forskolin (25μM final concentration diluted in dPBS), 25 μL diluted oxotremorine, and50 μL cells are added to agonist assay wells. To measure the ability ofa test compound to block oxotremorine-inhibited AC activity, 25 μLforskolin and oxotremorine (25 μM and 5 μM final concentrations,respectively, diluted in dPBS) 25 μL diluted test compound, and 50 μLcells are added to remaining assay wells.

Reactions are incubated for 10 minutes at 37° C. and stopped by additionof 100 μL ice-cold detection buffer. Plates are sealed, incubatedovernight at room temperature and counted the next morning on aPerkinElmer TopCount liquid scintillation counter (PerkinElmer Inc.,Wellesley, Mass.). The amount of cAMP produced (pmol/well) is calculatedbased on the counts observed for the samples and cAMP standards, asdescribed in the manufacturer's user manual. Data are analyzed bynonlinear regression analysis with the GraphPad Prism Software package(GraphPad Software, Inc., San Diego, Calif.) using the non-linearregression, one-site competition equation. The Cheng-Prusoff equation isused to calculate the K_(i), using the EC₅₀ of the oxotremorineconcentration-response curve and the oxotremorine assay concentration asthe K_(D) and [L], respectively. The K_(i) values are converted topK_(i) values to determine the geometric mean and 95% confidenceintervals. These summary statistics are then converted back to K_(i)values for data reporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. The exemplifiedcompounds of this invention are expected to have a K_(i) value of lessthan about 100 nM for blockade of oxotremorine-inhibition offorskolin-mediated cAMP accumulation in CHO-K1 cells expressing the hM₂receptor.

B. Blockade of Agonist-Mediated [³⁵S]GTPγS Binding

In a second functional assay, the functional potency of test compoundscan be determined by measuring the ability of the compounds to blockoxotremorine-stimulated [³⁵S]GTPγS binding in CHO-K1 cells expressingthe hM₂ receptor.

At the time of use, frozen membranes are thawed and then diluted inassay buffer with a final target tissue concentration of 5-10 μg proteinper well. The membranes are briefly homogenized using a Polytron PT-2100tissue disrupter and then added to the assay plates.

The EC₉₀ value (effective concentration for 90% maximal response) forstimulation of [³⁵S]GTPγS binding by the agonist oxotremorine isdetermined in each experiment.

To determine the ability of a test compound to inhibitoxotremorine-stimulated [³⁵S]GTPγS binding, the following is added toeach well of 96 well plates: 25 μL of assay buffer with [³⁵S]GTPγS (0.4nM), 25 μL of oxotremorine(EC₉₀) and GDP (3 μM), 25 μL of diluted testcompound and 25 μL CHO cell membranes expressing the hM₂ receptor. Theassay plates are then incubated at 37° C. for 60 minutes. The assayplates are filtered over 1% BSA-pretreated GF/B filters using aPerkinElmer 96-well harvester. The plates are rinsed with ice-cold washbuffer for 3×3 seconds and then air or vacuum dried. Microscint-20scintillation liquid (50 μL) is added to each well, and each plate issealed and radioactivity counted on a topcounter (PerkinElmer). Data areanalyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using thenon-linear regression, one-site competition equation. The Cheng-Prusoffequation is used to calculate the K_(i), using the IC₅₀ values of theconcentration-response curve for the test compound and the oxotremorineconcentration in the assay as the K_(D) and [L], ligand concentration,respectively.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. The exemplifiedcompounds of this invention are expected to have a K_(i) value of lessthan about 100 nM for blockade of oxotremorine-stimulated [³⁵S]GTPγSbinding in CHO-K1 cells expressing the hM₂ receptor.

C. Blockade of Agonist-Mediated Calcium Release via FLIPR Assays

Muscarinic receptor subtypes (M₁, M₃ and M₅ receptors), which couple toG_(q) proteins, activate the phospholipase C (PLC) pathway upon agonistbinding to the receptor. As a result, activated PLC hydrolyzesphosphatyl inositol diphosphate (PIP₂) to diacylglycerol (DAG) andphosphatidyl-1,4,5-triphosphate (IP₃), which in turn generates calciumrelease from intracellular stores, i.e., endoplasmic and sarcoplasmicreticulum. The FLIPR (Molecular Devices, Sunnyvale, Calif.) assaycapitalizes on this increase in intracellular calcium by using a calciumsensitive dye (Fluo-4AM, Molecular Probes, Eugene, Oreg.) thatfluoresces when free calcium binds. This fluorescence event is measuredin real time by the FLIPR, which detects the change in fluorescence froma monolayer of cells cloned with human M₁ and M₃, and chimpanzee M₅receptors. Antagonist potency can be determined by the ability ofantagonists to inhibit agonist-mediated increases in intracellularcalcium.

For FLIPR calcium stimulation assays, CHO cells stably expressing thehM₁, hM₃ and cM₅ receptors are seeded into 96-well FLIPR plates thenight before the assay is done. Seeded cells are washed twice byCellwash (MTX Labsystems, Inc.) with FLIPR buffer (10 mM HEPES, pH 7.4,2 mM calcium chloride, 2.5 mM probenecid in Hank's Buffered SaltSolution (HBSS) without calcium and magnesium) to remove growth mediaand leaving 50 μL/well of FLIPR buffer. The cells are then incubatedwith 50 μL/well of 4 μM FLUO-4AM (a 2× solution was made) for 40 minutesat 37° C., 5% carbon dioxide. Following the dye incubation period, cellsare washed two times with FLIPR buffer, leaving a final volume of 50μL/well.

To determine antagonist potency, the dose-dependent stimulation ofintracellular Ca²⁺ release for oxotremorine is first determined so thatantagonist potency can later be measured against oxotremorinestimulation at an EC₉₀ concentration. Cells are first incubated withcompound dilution buffer for 20 minutes, followed by agonist addition,which is performed by the FLIPR. An EC₉₀ value for oxotremorine isgenerated according to the method detailed in the FLIPR measurement anddata reduction section below, in conjunction with the formulaEC_(F)=((F/100−F)^1/H)*EC₅₀. An oxotremorine concentration of 3×EC_(F)is prepared in stimulation plates such that an EC₉₀ concentration ofoxotremorine is added to each well in the antagonist inhibition assayplates.

The parameters used for the FLIPR are: exposure length of 0.4 seconds,laser strength of 0.5 watts, excitation wavelength of 488 nm, andemission wavelength of 550 nm. Baseline is determined by measuring thechange in fluorescence for 10 seconds prior to addition of agonist.Following agonist stimulation, the FLIPR continuously measured thechange of fluorescence every 0.5 to 1 second for 1.5 minutes to capturethe maximum fluorescence change.

The change of fluorescence is expressed as maximum fluorescence minusbaseline fluorescence for each well. The raw data is analyzed againstthe logarithm of drug concentration by nonlinear regression withGraphPad Prism (GraphPad Software, Inc., San Diego, Calif.) using thebuilt-in model for sigmoidal dose-response. Antagonist K_(i) values aredetermined by Prism using the oxotremorine EC₅₀ value as the K_(D) andthe oxotremorine EC₉₀ for the ligand concentration according to theCheng-Prusoff equation (Cheng & Prusoff, 1973).

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. The exemplifiedcompounds of this invention are expected to have a K_(i) value of lessthan about 100 nM for blockade of agonist-mediated calcium release inCHO cells stably expressing the hM₃ receptor.

Assay 3 Determination of Duration of Bronchoprotection in Guinea PigModel of Acetylcholine-Induced Bronchoconstriction

This in vivo assay was used to assess the bronchoprotective effects oftest compounds exhibiting muscarinic receptor antagonist activity.

Groups of six male guinea pigs (Duncan-Hartley (HsdPoc:DH) Harlan,Madison, Wis.) weighing between 250 and 350 g were individuallyidentified by cage cards. Throughout the study animals were allowedaccess to food and water ad libitum.

Test compounds were administered via inhalation over 10 minutes in awhole-body exposure dosing chamber (R&S Molds, San Carlos, Calif.). Thedosing chambers were arranged so that an aerosol was simultaneouslydelivered to 6 individual chambers from a central manifold. Guinea pigswere exposed to an aerosol of a test compound or vehicle (WFI). Theseaerosols were generated from aqueous solutions using an LC StarNebulizer Set (Model 22F51, PARI Respiratory Equipment, Inc. Midlothian,Va.) driven by a mixture of gases (CO₂=5%, O₂=21% and N₂=74%) at apressure of 22 psi. The gas flow through the nebulizer at this operatingpressure was approximately 3 L/minute. The generated aerosols weredriven into the chambers by positive pressure. No dilution air was usedduring the delivery of aerosolized solutions. During the 10 minutenebulization, approximately 1.8 mL of solution was nebulized. This wasmeasured gravimetrically by comparing pre-and post-nebulization weightsof the filled nebulizer.

The bronchoprotective effects of test compounds administered viainhalation were evaluated using whole body plethysmography at 1.5, 24,48 and 72 hours post-dose.

Forty-five minutes prior to the start of the pulmonary evaluation, eachguinea pig was anesthetized with an intramuscular injection of ketamine(43.75 mg/kg), xylazine (3.50 mg/kg) and acepromazine (1.05 mg/kg).After the surgical site was shaved and cleaned with 70% alcohol, a 2-3cm midline incision of the ventral aspect of the neck was made. Then,the jugular vein was isolated and cannulated with a saline-filledpolyethylene catheter (PE-50, Becton Dickinson, Sparks, Md.) to allowfor intravenous infusions of ACh (Sigma-Aldrich, St. Louis, Mo.) insaline. The trachea was then dissected free and cannulated with a 14 Gteflon tube (#NE-014, Small Parts, Miami Lakes, Fla.). If required,anesthesia was maintained by additional intramuscular injections of theaforementioned anesthetic mixture. The depth of anesthesia was monitoredand adjusted if the animal responded to pinching of its paw or if therespiration rate was greater than 100 breaths/minute.

Once the cannulations were complete, the animal was placed into aplethysmograph (#PLY3114, Buxco Electronics, Inc., Sharon, Conn.) and anesophageal pressure cannula (PE-160, Becton Dickinson, Sparks, Md.) wasinserted to measure pulmonary driving pressure (pressure). The teflontracheal tube was attached to the opening of the plethysmograph to allowthe guinea pig to breathe room air from outside the chamber. The chamberwas then sealed. A heating lamp was used to maintain body temperatureand the guinea pig's lungs were inflated 3 times with 4 mL of air usinga 10 mL calibration syringe (#5520 Series, Hans Rudolph, Kansas City,Mo.) to ensure that the lower airways did not collapse and that theanimal did not suffer from hyperventilation.

Once it was determined that baseline values were within the range0.3-0.9 mL/cm H₂O for compliance and within the range 0.1-0.199 cmH₂O/mL per second for resistance, the pulmonary evaluation wasinitiated. A Buxco pulmonary measurement computer progam enabled thecollection and derivation of pulmonary values.

Starting this program initiated the experimental protocol and datacollection. The changes in volume over time that occur within theplethysmograph with each breath were measured via a Buxco pressuretransducer. By integrating this signal over time, a measurement off lowwas calculated for each breath. This signal, together with the pulmonarydriving pressure changes, which were collected using a Sensym pressuretransducer (#TRD4100), was connected via a Buxco (MAX 2270) preamplifierto a data collection interface (#'s SFT3400 and SFT3813). All otherpulmonary parameters were derived from these two inputs.

Baseline values were collected for 5 minutes, after which time theguinea pigs were challenged with ACh. ACh (0.1 mg/mL) was infusedintravenously for 1 minute from a syringe pump (sp210iw, World PrecisionInstruments, Inc., Sarasota, Fla.) at the following doses and prescribedtimes from the start of the experiment: 1.9 μg/minute at 5 minutes, 3.8μg/minute at 10 minutes, 7.5 μg/minute at 15 minutes, 15.0 μg/minute at20 minutes, 30 μg/minute at 25 minutes and 60 μg/minute at 30 minutes.If resistance or compliance had not returned to baseline values at 3minutes following each ACh dose, the guinea pig's lungs were inflated 3times with 4 mL of air from a 10 mL calibration syringe. Recordedpulmonary parameters included respiration frequency (breaths/minute),compliance (mL/cm H₂O) and pulmonary resistance (cm H₂O/mL per second).Once the pulmonary function measurements were completed at minute 35 ofthis protocol, the guinea pig was removed from the plethysmograph andeuthanized by carbon dioxide asphyxiation.

The data were evaluated in one or both of the following ways:

(a) Pulmonary resistance (R_(L), cm H₂O/mL per second) was calculatedfrom the ratio of “change in pressure” to “the change in flow.” TheR_(L) response to ACh (60 μg/min, IH) was computed for the vehicle andthe test compound groups. The mean ACh response in vehicle-treatedanimals, at each pre-treatment time, was calculated and used to compute% inhibition of ACh response, at the corresponding pre-treatment time,at each test compound dose. Inhibition dose-response curves for ‘R_(L)’were fitted with a four parameter logistic equation using GraphPadPrism, version 3.00 for Windows (GraphPad Software, San Diego, Calif.)to estimate bronchoprotective ID₅₀ (dose required to inhibit the ACh (60μg/min) bronchoconstrictor response by 50%). The equation used was asfollows:Y=Min+(Max−Min)/(1+10^(((log ID50−X)*Hillslope)))where X is the logarithm of dose, Y is the response (% Inhibition of AChinduced increase in R_(L)). Y starts at Min and approachesasymptotically to Max with a sigmoidal shape.

(b) The quantity PD₂, which is defined as the amount of ACh or histamineneeded to cause a doubling of the baseline pulmonary resistance, wascalculated using the pulmonary resistance values derived from the flowand the pressure over a range of ACh or histamine challenges using thefollowing equation (which is derived from a equation used to calculatePC₂₀ values described in American Thoracic Society. Guidelines formethacholine and exercise challenge testing—1999. Am J Respir Crit CareMed. 161: 309-329 (2000)):

${PD}_{2} = {{antilog}\mspace{14mu}\left\lbrack {{\log\mspace{14mu} C_{1}} + \frac{\left( {{\log\mspace{14mu} C_{2}} - {\log\mspace{14mu} C_{1}}} \right)\left( {{2R_{0}} - R_{1}} \right)}{R_{2} - R_{1}}} \right\rbrack}$where:

-   -   C₁=concentration of ACh or histamine preceding C₂    -   C₂=concentration of ACh or histamine resulting in at least a        2-fold increase in pulmonary resistance (R_(L))    -   R₀=Baseline R_(L) value    -   R₁=R_(L) value after C₁    -   R₂=R_(L) value after C₂

An efficacious dose was defined as a dose that limited thebronchrestriction response to a 50 μg/mL dose of ACh to a doubling ofthe baseline pulmonary resistance (PD₂₍₅₀₎). Statistical analysis of thedata was performed using a two-tailed Students t-test. A P-value<0.05was considered significant.

Generally, test compounds having a PD₂₍₅₀₎ less than about 300 μg/mL forACh-induced bronchoconstriction at 1.5 hours post-dose in this assay arepreferred. For example, the compounds of Examples 2 and 3 were found tohave a PD₂₍₅₀₎ less than about 300 μg/mL for ACh-inducedbronchoconstriction at 1.5 hours post-dose.

Assay 4 Inhalation Guinea Pig Salivation Assay

Guinea pigs (Charles River, Wilmington, Mass.) weighing 200-350 g wereacclimated to the in-house guinea pig colony for at least 3 daysfollowing arrival. Test compound or vehicle were dosed via inhalation(IH) over a 10 minute time period in a pie shaped dosing chamber (R&SMolds, San Carlos, Calif.). Test solutions were dissolved in sterilewater and delivered using a nebulizer filled with 5.0 mL of dosingsolution. Guinea pigs were restrained in the inhalation chamber for 30minutes. During this time, guinea pigs were restricted to an area ofapproximately 110 sq. cm. This space was adequate for the animals toturn freely, reposition themselves, and allow for grooming. Following 20minutes of acclimation, guinea pigs were exposed to an aerosol generatedfrom a LS Star Nebulizer Set (Model 22F51, PARI Respiratory Equipment,Inc. Midlothian, Va.) driven by house air at a pressure of 22 psi. Uponcompletion of nebulization, guinea pigs were evaluated at 1.5, 6, 12,24, 48, or 72 hrs after treatment.

Guinea pigs were anesthetized one hour before testing with anintramuscular (IM) injection of a mixture of ketamine 43.75 mg/kg,xylazine 3.5 mg/kg, and acepromazine 1.05 mg/kg at an 0.88 mL/kg volume.Animals were placed ventral side up on a heated (37° C.) blanket at a 20degree incline with their head in a downward slope. A 4-ply 2×2 inchgauze pad (Nu-Gauze General-use sponges, Johnson and Johnson, Arlington,Tex.) was inserted in the guinea pig's mouth. Five minutes later, themuscarinic agonist pilocarpine (3.0 mg/kg, SC) was administered and thegauze pad was immediately discarded and replaced by a new pre-weighedgauze pad. Saliva was collected for 10 minutes, at which point the gauzepad was weighed and the difference in weight recorded to determine theamount of accumulated saliva (in mg). The mean amount of salivacollected for animals receiving the vehicle and each dose of testcompound was calculated. The vehicle group mean was considered to be100% salivation. Results were calculated using result means (n=3 orgreater). Confidence intervals (95%) were calculated for each dose ateach time point using two-way ANOVA. This model is a modified version ofthe procedure described in Rechter, “Estimation of anticholinergic drugeffects in mice by antagonism against pilocarpine-induced salivation”Ata Pharmacol Toxicol 24:243-254 (1996)

The mean weight of saliva in vehicle-treated animals, at eachpre-treatment time, was calculated and used to compute % inhibition ofsalivation, at the corresponding pre-treatment time, at each dose. Theinhibition dose-response data were fitted to a four parameter logisticequation using GraphPad Prism, version 3.00 for Windows (GraphPadSoftware, San Diego, Calif.) to estimate anti-sialagogue ID₅₀ (doserequired to inhibit 50% of pilocarpine-evoked salivation). The equationused was as follows:Y=Min+(Max−Min)/(1+10^(((log ID50−X)*Hillslope)))where X is the logarithm of dose, Y is the response (% inhibition ofsalivation). Y starts at Min and approaches asymptotically to Max with asigmoidal shape.

The ratio of the anti-sialagogue ID₅₀ to bronchoprotective ID₅₀ was usedto compute the apparent lung selectivity index of the test compound.Generally, compounds having an apparent lung selectivity index greaterthan about 5 are preferred. In this assay, the compound of Example 2 hadan apparent lung-selectivity index greater than about 15.

Assay 5 Methacholine-Induced Depressor Responses in Conscious GuineaPigs

Healthy, adult, male Sprague-Dawley guinea pigs (Harlan, Indianapolis,Ind.), weighing between 200 and 300 g are used in these studies. Underisoflurane anesthesia (to effect), animals are instrumented with commoncarotid artery and jugular vein catheters (PE-50 tubing). The cathetersare exteriorized utilizing a subcutaneous funnel to the subscapulararea. All surgical incisions are sutured with 4-0 Ethicon Silk and thecatheters locked with heparin (1000 units/mL). Each animal isadministered saline (3 mL, SC) at the end of surgery as well asbuprenorphine (0.05 mg/kg, IM). Animals are allowed to recover on aheating pad before being returned to their holding rooms.

Approximately 18 to 20 hours following surgery, the animals are weighedand the carotid artery catheter on each animal is connected to atransducer for recording arterial pressure. Arterial pressure and heartrate is recorded using a Biopac MP-100 Acquisition System. Animals areallowed to acclimate and stabilize for a period of 20 minutes.

Each animal is challenged with MCh (0.3 mg/kg, IV) administered throughthe jugular venous line and the cardiovascular response is monitored for10 minutes. The animals are then placed into the whole body dosingchamber, which is connected to a nebulizer containing the test compoundor vehicle solution. The solution is nebulized for 10 minutes using agas mixture of breathable air and 5% carbon dioxide with a flow rate of3 liters/minute. The animals are then removed from the whole bodychamber and returned to their respective cages. At 1.5 and 24 hpost-dosing, the animals are re-challenged with MCh (0.3 mg/kg, IV) andthe hemodynamic response is determined. Thereafter, the animals areeuthanized with sodium pentobarbital (150 mg/kg, IV).

MCh produces a decrease in mean arterial pressure (MAP) and decrease inheart rate (bradycardia). The peak decrease, from baseline, in MAP(depressor responses) was measured for each MCh challenge (before andafter IH dosing). The bradycardic effects are not used for analysissince these responses are not robust and reproducible. The effects oftreatment on the MCh responses are expressed as % inhibition(mean+/−SEM) of the control depressor responses. Two-way ANOVA with theappropriate post-hoc test was used to test the effects of treatment andpre-treatment time. The depressor responses to MCh were relativelyunchanged at 1.5 and 24 h after inhalation dosing with vehicle.

The ratio of the anti-depressor ID₅₀ to bronchoprotective ID₅₀ was usedto compute apparent lung-selectivity of the test compound. Generally,compounds having an apparent lung-selectivity index greater than 5 arepreferred. In this assay, compounds of the invention are expected tohave an apparent lung-selectivity index greater than 5.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statues and regulations, all publications, patents andpatent applications cited herein are hereby incorporated by reference intheir entirety to the same extent as if each document had beenindividually incorporated by reference herein.

1. A compound of formula I:

wherein: each R¹ and R² are independently selected from (1-4C)alkyl,(2-4C)alkenyl, (2-4C)alkynyl, (3-6C)cycloalkyl, cyano, halo, —OR^(a),—SR^(a), —NR^(a)R^(b), —S(O)R^(c) and —S(O)₂R^(c); where each R^(a) andR^(b) independently represents hydrogen, (1-4C)alkyl, (2-4C)alkenyl,(2-4C)alkynyl or (3-6C)cycloalkyl; each R^(c) independently represents(1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl or (3-6C)cycloalkyl; or twoadjacent R¹ groups or two adjacent R² groups are joined together to form(3-6C)alkylene, (2-4C)alkylene-O— or —O—(2-4C)alkylene-O—; a and b eachindependently are 0 or an integer of from 1 to 5; each R³ independentlyis fluoro or (1-4C)alkyl; c is 0 or an integer of from 1 to 3; R^(4a)and R^(4b) are independently selected from hydrogen, (1-4C)alkyl andphenyl-(1-4C)alkyl; or R^(4a) and R^(4b) together with the carbon atomto which they are attached form a (3-6C)heterocyclic ring optionallycontaining one additional heteroatom selected from nitrogen, oxygen orsulfur and wherein the heterocyclic ring is unsubstituted or substitutedwith 1 or 2 substituents selected independently from (1-4C)alkyl andfluoro; e is 1; m is 1, 2, 3 or 4; Ar¹ represents a phenylene group or a(3-5C)heteroarylene group containing 1 or 2 heteroatoms selectedindependently from oxygen, nitrogen or sulfur; wherein the phenylene andheteroarylene groups are unsubstituted or substituted with 1 to 4substituents selected independently from halo, (1-4C)alkyl or(1-4C)alkoxy; wherein each alkyl and alkoxy group is optionallysubstituted with from 1 to 3 fluoro substituents; n is 0, 1, 2, 3 or 4;provided that the values of m, n and Ar¹ are selected such that thenumber of contiguous atoms in the chain —(CH₂)_(m)—Ar¹—(CH₂)_(n)—between the two nitrogen atoms to which it is attached is in the rangeof from 7 to 12; R⁵ is selected from hydrogen, (1-6C)alkyl, Ar², —CH₂Ar² and —CH₂CH₂NHC(O)R^(5a); where Ar² represents phenyl,(3-6C)cycloalkyl or (3-5C)heteroaryl containing 1 or 2 heteroatomsselected from oxygen, nitrogen or sulfur, wherein the phenyl andheteroaryl groups are unsubstituted or substituted with 1 to 3substituents selected independently from halo, (1-4C)alkyl, (1-4C)alkoxyand methylenedioxy; and wherein R^(5a) represents (1-4C)alkyl; R⁶ ishydrogen or (1-6C)alkyl; or R⁵ and R⁶ together with the nitrogen atom towhich they are attached form a (3-5C)azacycloalkyl group; or when Ar¹represents heteroarylene, R⁵ and R⁶ together with the nitrogen atom towhich they are attached can additionally form a morpholin-1-yl or4-(1-6C)alkylpiperazin-1-yl group; and wherein each alkyl group in R¹,R², R³, R^(4a), R^(4b), R⁵, R⁶ and R^(a-c) is optionally substitutedwith from 1 to 5 fluoro substituents; or a pharmaceutically acceptablesalt or solvate or stereoisomer thereof.
 2. The compound of claim 1,wherein a, b and c each represents
 0. 3. The compound of claim 1,wherein R^(4a) and R^(4b) are hydrogen.
 4. The compound of claim 1,wherein m is 2, 3 or
 4. 5. The compound of claim 1, wherein n is 0, 2 or3.
 6. The compound of any one of claims 1 to 5, wherein Ar¹ represents1,3-phenylene, 1,4-phenylene or 2,5-thienylene; wherein the phenylene orthienylene group is unsubstituted or substituted.
 7. The compound of anyone of claims 1 to 5, wherein R⁵ is (1-4C)alkyl.
 8. The compound of anyone of claims 1 to 5, wherein R⁶ is hydrogen or methyl.
 9. The compoundof claim 1, where a, b, and c are 0; R^(4a) and R^(4b) are hydrogen; Ar¹is phen-1,4-ylene, unsubstituted or substituted with 1 to 4 substituentsselected independently from halo, (1-4C)alkyl or (1-4C)alkoxy; whereineach alkyl and alkoxy group is optionally substituted with from 1 to 3fluoro substituents; and m+n is an integer from 3 to
 8. 10. The compoundof claim 9, wherein m is 4 and n is 0, or m is 2 and n is 2, or m is 3and n is
 2. 11. The compound of claim 1, where a, b, and c are 0; R^(4a)and R^(4b) are hydrogen; Ar¹ is phen-1,3-ylene, unsubstituted orsubstituted with 1 to 4 substituents selected independently from halo,(1-4C)alkyl or (1-4C)alkoxy; wherein each alkyl and alkoxy group isoptionally substituted with from 1 to 3 fluoro substituents; and m+n isan integer from 4 to
 9. 12. The compound of claim 11, wherein m is 2 andn is 4, or m is 3 and n is
 3. 13. The compound of claim 1, where a, b,and c are 0; R^(4a) and R^(4b) are hydrogen; Ar¹ is 2,5-thiophene,unsubstituted or substituted with 1 to 2 substituents selectedindependently from halo, (1-4C)alkyl or (1-4C)alkoxy; wherein each alkyland alkoxy group is optionally substituted with from 1 to 3 fluorosubstituents; and m+n is an integer from 4 to
 9. 14. The compound ofclaim 13, wherein m is 3 and n is
 3. 15. The compound of claim 13,wherein R⁵ and R⁶ together with the nitrogen atom to which they areattached form a morpholin-1-yl or 4-(1-6C)alkylpiperazin-1-yl group. 16.The compound of any one of claims 10, 12 and 14, where R⁵ is selectedfrom hydrogen; (1-4C)alkyl; —CH₂Ar² where Ar² represents phenyl,furan-2-yl, 3,4-methylenedioxyphenyl, or 5-methylpyrazin-2-yl; and—CH₂CH₂NHCOR^(5a), where R^(5a) is methyl.
 17. The compound of any oneof claims 9, 11 and 13, where R⁶ is hydrogen or methyl.
 18. The compoundof any one of claims 9, 11 and 13, where R⁵ and R⁶ together with thenitrogen atom to which they are attached form a (3-4C)azacycloalkylgroup.
 19. A compound selected from:2-{(S)-1-[4-(4-aminophenyl)butyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-((S)-1-{2-[4-(2-methylaminoethyl)phenyl]ethyl}pyrrolidin-3-yl)-2,2-diphenylacetamide;2-((S)-1-{2-[3-(2-methylaminoethyl)phenyl]ethyl}-pyrrolidin-3-yl)-2,2-diphenylacetamide;2-((S)-1-{3-[5-(3-methylaminopropyl)thiophen-2-yl]propyl}-pyrrolidin-3-yl)-2,2-diphenylacetamide;2-((S)-1-{3-[3-(3-methylaminopropyl)phenyl]propyl}-pyrrolidin-3-yl)-2,2-diphenylacetamide;2-((S)-1-{3-[4-(2-methylaminoethyl)phenyl]propyl}-pyrrolidin-3-yl)-2,2-diphenylacetamide;2-[(S)-1-{3-[5-(3-ethylaminopropyl)thiophen-2-yl]propyl}-pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(S)-1-{3-[5-(3-isopropylaminopropyl)thiophen-2-yl]propyl}-pyrrolidin-3-y]-2,2-diphenylacetamide;2-[(S)-1-{3-[5-(3-dimethylaminopropyl)thiophen-2-yl]-propyl}pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(S)-1-{2-[4-(2-ethylaminoethyl)phenyl]ethyl}pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(S)-1-{2-[4-(2-isopropylaminoethyl)phenyl]ethyl}pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(S)-1-{2-[4-(2-dimethylaminoethyl)phenyl]ethyl}pyrrolidin-3-yl]-2,2-diphenylacetamide;2,2-diphenyl-2-[(S)-1-{2-[4-(2-pyrrolidin-1-ylethyl)phenyl]ethyl}pyrrolidin-3-yl]acetamide;2-[(S)-1-(2-{4-[2-(benzylmethylamino)ethyl]phenyl}ethyl)-pyrrolidin-3-yl]-2,2-diphenylacetamide;2-[(S)-1-(2-{4-[2-(2-acetylaminoethylamino)ethyl]phenyl}ethyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-{(S)-1-[2-(4-{2-[(5-methylpyrazin-2-ylmethyl)amino]ethyl}-phenyl)ethyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(S)-1-[2-(4-{2-[(furan-2-ylmethyl)amino]ethyl}phenyl)ethyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(S)-1-[2-(4-{2-[(benzo[1,3]dioxol-5-ylmethyl)amino]ethyl}-phenyl)ethyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-((S)-1-{2-[4-(2-azetidin-1-ylethyl)phenyl]ethyl}pyrrolidin-3-yl)-2,2-diphenylacetamide,2-[(S)-1-(3-{5-[3-(2-acetylaminoethylamino)propyl]thiophen-2-yl}-propyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-{(S)-1-[3-(5-{3-[(5-methylpyrazin-2-ylmethyl)amino]propyl}thiophen-2-yl)propyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(S)-1-[3-(5-{3-[(furan-2-ylmethyl)amino]propyl}thiophen-2-yl)-propyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(S)-1-[3-(5-{3-[(benzo[1,3]dioxol-5-ylmethyl)amino]propyl}thiophen-2-yl)propyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-[(S)-1-(3-{5-[3-(4-methylpiperazin-1-yl)propyl]thiophen-2-yl}propyl)pyrrolidin-3-yl]-2,2-diphenylacetamide;2-((S)-1-{3-[5-(3-morpholin-4-yl-propyl)thiophen-2-yl]propyl}pyrrolidin-3-yl)-2,2-diphenylacetamide;and2-((S)-1-{3-[5-(3-azetidin-1-ylpropyl)thiophen-2-yl]propyl}pyrrolidin-3-yl)-2,2-diphenylacetamide;or a pharmaceutically acceptable salt or solvate thereof.
 20. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of a compound of claim 1or
 19. 21. The pharmaceutical composition of claim 20, wherein thecomposition further comprises a therapeutically effective amount of anagent selected from β₂ adrenergic receptor agonists, steroidalanti-inflammatory agents, phosphodiesterase-4 inhibitors, andcombinations thereof.
 22. The pharmaceutical composition of claim 21,wherein the composition comprises a therapeutically effective amount ofa β₂ adrenergic receptor agonist and a steroidal anti-inflammatoryagent.
 23. A process for preparing a compound of claim 1 or 19, theprocess comprising: (a) reacting a compound of formula II:

with a compound of formula III:X¹—(CH₂)_(m)—Ar¹—(CH₂)_(n)—NR⁵R⁶  III wherein X¹ represents a leavinggroup; (b) reacting a compound of formula IV:

with a compound of formula V:HNR⁵R⁶  V in the presence of a reducing agent; (c) reacting a compoundof formula VI:

wherein X² represents a leaving group, with a compound of formula V; (d)reacting a compound of formula II with a compound of formula VII:OHC—(CH₂)_(m-1)—Ar¹—(CH₂)_(n)—NR⁵R⁶  VII in the presence of a reducingagent; or (e) reacting a compound of formula VIII:

with a reducing agent; to provide a compound of formula I.
 24. Theprocess of claim 23, wherein the process further comprises forming apharmaceutically acceptable salt of the compound of formula I.
 25. Amethod of treating chronic obstructive pulmonary disease or asthma, themethod comprising administering to a patient a therapeutically effectiveamount of a compound of claim 1 or 19.